Collaborative orchestration of BH3-only proteins governs Bak/Bax-dependent hepatocyte apoptosis under antiapoptotic protein-deficiency in mice

Collaborative orchestration of BH3-only proteins governs Bak/Bax-dependent hepatocyte apoptosis under antiapoptotic protein-deficiency in mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Collaborative orchestration of BH3-only proteins governs Bak/Bax-dependent hepatocyte apoptosis under antiapoptotic protein-deficiency in mice Tetsuo Takehara, Shinnosuke Kudo, Hayato Hikita, Yoshinobu Saito, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4704982/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Feb, 2025 Read the published version in Cell Death & Differentiation → Version 1 posted 11 You are reading this latest preprint version Abstract The fine-tuned balance between anti-apoptotic Bcl-2 family proteins, such as Bcl-xL and Mcl-1, and pro-apoptotic Bcl-2 family proteins, like Bak and Bax, is crucial for maintaining hepatocyte integrity. BH3-only proteins, including Bid, Bim, Puma, Noxa, Bad, Bik, Bmf and Hrk, serve as apoptosis sensors activating Bak and Bax. We previously reported that BH3-only proteins Bid and Bim contribute to hepatocyte apoptosis through Bak/Bax activation in the absence of antiapoptotic proteins, Bcl-xL and/or Mcl-1. However, the comprehensive involvement of all eight BH3-only proteins maintaining hepatocyte integrity in healthy livers remains unclear. Puma disruption suppressed hepatocyte apoptosis in hepatocyte-specific Bcl-xL or Mcl-1 knockout (Bcl-xL ΔHep/ΔHep or Mcl-1 ΔHep/ΔHep ) mice. Disruption of Bid and Bim partially prevented lethality in Mcl-1 ΔHep/+ Bcl-xL ΔHep/ΔHep mice, although severe hepatocyte apoptosis persisted, which was suppressed by additional Puma disruption. However, hepatocyte apoptosis was still strongly induced compared to that in Mcl-1 ΔHep/+ Bcl-xL ΔHep/ΔHep Bax ΔHep/ΔHep Bak −/− mice. Triple disruption of Bid, Bim and Puma did not prevent induction of hepatocyte apoptosis in tamoxifen-induced Mcl-1 iΔHep/iΔHep Bcl-xL iΔHep/iΔHep mice. Primary hepatocytes, isolated from Mcl-1 fl/fl Bcl-xL fl/fl Bid −/− Bim −/− Puma −/− mice and immortalized, underwent apoptosis with doxycycline-dependent Cre recombination. Among the remaining five BH3-only proteins, Bik and Hrk were not expressed in this cell line, and Noxa knockdown, but not Bad or Bmf knockdown, reduced apoptosis. Noxa disruption alleviated hepatocyte apoptosis in Mcl-1 ΔHep/ΔHep mice and tamoxifen-induced Mcl-1 iΔHep/iΔHep Bcl-xL iΔHep/iΔHep Bid −/− Bim −/− Puma −/− mice, prolonging survival. Apoptosis persisted in immortalized primary hepatocytes isolated from Mcl-1 fl/fl Bcl-xL fl/fl Bid −/− Bim −/− Puma −/− Noxa −/− mice where doxycycline-dependent Cre recombination was induced, but was completely suppressed by Bak/Bax knockdown, while Bad or Bmf knockdown had no effect. In conclusion, among the eight BH3-only proteins, Puma and Noxa, alongside Bid and Bim, contributed to sustained Bak/Bax-dependent hepatocyte apoptosis in the absence of Mcl-1 and Bcl-xL, elucidating the orchestration of Bcl-2 family proteins in healthy livers. Biological sciences/Cell biology Biological sciences/Biochemistry/Proteins Biological sciences/Physiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The mitochondrial pathway of hepatocyte apoptosis is orchestrated by members of B-cell lymphoma-2 (Bcl-2) family proteins, which include the anti-apoptotic proteins Bcl-2, B-cell lymphoma-extra large (Bcl-xL), myeloid cell leukemia-1 (Mcl-1), B-cell lymphoma-w (Bcl-w), and Bcl-2-related protein A1 (Bcl2A1), as well as the pro-apoptotic proteins Bcl-2-antagonist/killer (Bak) and Bcl-2-associated X protein (Bax) 1 . Pro-apoptotic proteins act as effector molecules in this apoptotic cascade 2 . Upon activation, they form pores in the mitochondrial outer membrane, facilitating the release of cytochrome c. This leads to the activation of the caspase cascade and finally leads to apoptosis 3 . Anti-apoptotic Bcl-2 family members, including Bcl-xL and Mcl-1, inhibit the mitochondrial pathway of apoptosis by neutralizing Bak/Bax activity. Bcl-2 homology domain 3 (BH3)-only proteins, a group of eight members, Bid, Bim, Puma, Noxa, Bad, Bmf, Bik and Hrk, serve as sensor proteins of apoptosis, and they induce apoptosis by activating Bak/Bax 4 . In healthy hepatocytes, Bcl-xL and Mcl-1 are crucial antiapoptotic proteins, and we previously reported that hepatocyte-specific Mcl-1 knockout (KO) ( Mcl-1 flox/flox Alb-Cre ) mice (Mcl-1 ΔHep/ΔHep mice) and hepatocyte-specific Bcl-xL KO ( Bcl-xL flox/flox Alb-Cre ) mice (Bcl-xL ΔHep/ΔHep mice) exhibit spontaneous hepatocyte apoptosis 5 , 6 . Moreover, knockout of Bak or Bax abolished hepatocyte apoptosis in these mice 5 , 7 , suggesting that hepatocyte apoptosis induced by deletion of Mcl-1 or Bcl-xL causes autoactivation of Bak/Bax and leads to hepatocyte apoptosis 8 . We previously reported that among the eight BH3-only proteins Bid and Bim were functionally active and that disruption of Bid and Bim decreased hepatocyte apoptosis in Bcl-xL ΔHep/ΔHep mice and Mcl-1 ΔHep/ΔHep mice 9 . However, the involvement of all eight BH3-only proteins in maintaining hepatocyte integrity in the healthy liver remains to be elucidated. In the present study, we investigated the comprehensive involvement of all eight BH3-only proteins in Bak/Bax-dependent hepatocyte apoptosis in the absence of antiapoptotic Bcl-2 family proteins. We demonstrated that among the eight BH3-only proteins, Puma and Noxa, together with Bid and Bim, are essential regulators that promote Bak/Bax-dependent hepatocyte apoptosis in vivo. Our present study revealed the orchestrated role of BH3-only proteins in maintaining hepatocyte integrity in the healthy liver via Bak/Bax-dependent apoptosis. Results Disruption of Puma suppresses hepatocyte apoptosis in mice with hepatocyte-specific knockout of Bcl-xL or Mcl-1. We initially examined the role of another BH3-only protein, Puma, in Bak/Bax activation in the absence of Bcl-xL or Mcl-1. We crossed Mcl-1 ΔHep/ΔHep mice and Bcl-xL ΔHep/ΔHep mice with Puma KO ( Puma −/− ) mice (Puma −/− mice), and generated Mcl-1 ΔHep/Δhep Puma −/− mice and Bcl-xL Δhep/Δhep Puma −/− mice. The expression levels of Mcl-1 and Puma were reduced in the liver tissue of Mcl-1 ΔHep/ΔHep Puma −/− mice (Fig. 1 A). Similarly, Bcl-xL and Puma expression in the livers of Bcl-xL ΔHep/ΔHep Puma −/− mice was reduced (Fig. 1 B). Mcl-1 ΔHep/ΔHep Puma −/− mice and Bcl-xL ΔHep/ΔHep Puma −/− mice displayed significantly lower serum alanine transaminase (ALT) levels and reduced serum caspase 3/7 activity than Mcl-1 ΔHep/ΔHep mice and Bcl-xL ΔHep/ΔHep mice, respectively (Fig. 1 C and 1 D). These mice also showed a smaller number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive hepatocytes. (Fig. 1 E and 1 F). Disruption of Bid, Bim and Puma cannot fully suppress hepatocyte apoptosis induced by the deletion of Bcl-xL and Mcl-1. We have previously demonstrated that Bcl-xL ΔHep/ΔHep Mcl-1 ΔHep/+ mice, as well as Bcl-xL ΔHep/ΔHep Mcl-1 ΔHep/ΔHep mice, exhibit impaired liver development, leading to mortality within one day after birth 10 . Disruption of both Bid and Bim or Bid, Bim and Puma reduced the mortality of Bcl-xL ΔHep/ΔHep Mcl-1 ΔHep/+ mice but had no effect on that of Bcl-xL ΔHep/ΔHep Mcl-1 ΔHep/ ΔHep mice ( Table 1, Table 2 ). Surviving Bcl-xL ΔHep/ΔHep Mcl-1 ΔHep/+ Bid −/− Bim −/− mice exhibited hepatocyte apoptosis with elevated serum ALT levels (Fig. 2 A- 2 D). Further disruption of Puma significantly reduced the serum ALT levels, serum caspase 3/7 activity and number of TUNEL-positive hepatocytes in Bcl-xL ΔHep/ΔHep Mcl-1 ΔHep/+ Bid −/− Bim −/− mice (Fig. 2 B- 2 D). However, the serum ALT levels in Bcl-xL ΔHep/ΔHep Mcl-1 ΔHep/+ Bid −/− Bim −/− Puma −/− mice were still significantly greater than those in Bcl-xL ΔHep/ΔHep Mcl-1 ΔHep/+ Bax ΔHep/ΔHep Bak −/− mice (Fig. 2 B). We generated tamoxifen-inducible Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bid −/− Bim −/− Puma −/− mice, in which Bcl-xL and Mcl-1 expression are abolished upon tamoxifen injection (Fig. 2 E), since the knockout of both Bcl-xL and Mcl-1 severely impairs liver development 10 and no Bcl-xL ΔHep/ΔHep Mcl-1 ΔHep/ΔHep Bid −/− Bim −/− Puma −/− mice survive after birth ( Table 2 ). After tamoxifen injection for three consecutive days, compared with control mice, Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bid −/− Bim −/− Puma −/− mice exhibited increased serum ALT levels, elevated caspase 3/7 activity and a higher number of TUNEL-positive hepatocytes (Fig. 2 F-H). All mice in the Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bid −/− Bim −/− Puma −/− group were euthanized within 4 days after tamoxifen injection (data not shown). On the other hand, Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bax iΔHep/iΔHep Bak −/− mice showed no increase in serum ALT levels after tamoxifen injection compared to those without tamoxifen injection ( Suppl. Figure 1A, 1B) , and all of these mice survived to day 6 after tamoxifen injection ( Suppl. Figure 1C) . These findings suggested that another BH3-only protein, in addition to Bid, Bim and Puma, might activate Bak/Bax and induce hepatocyte apoptosis in the absence of both Bcl-xL and Mcl-1. Noxa knockdown suppresses apoptosis in Bcl-xL-, Mcl-1-, Bid-, Bim- and Puma-deficient hepatocytes. We generated a doxycycline-inducible Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bid −/− Bim −/− Puma −/− hepatocyte cell line using immortalized primary mouse hepatocytes whose Bcl-xL and Mcl-1 expression was abolished 48 hours after incubation with doxycycline (Fig. 3 A). Caspase 3/7 activity and LDH activities were significantly increased, and the relative cell viability was significantly decreased after incubation with doxycycline (Fig. 3 B). Among the five remaining BH3-only proteins, Noxa , Bad , and Bmf were expressed in this immortalized cell line, while Bik and Hrk were not detected (Fig. 3 C). After doxycycline treatment, only Noxa expression increased among Noxa, Bad and Bmf (Fig. 3 D). SiRNA-mediated knockdown of Noxa led to a significant reduction in Caspase 3/7 activity, LDH activities, and Annexin V-positive areas and a significant increase in the relative cell viability in doxycycline-treated Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bid −/− Bim −/− Puma −/− cells (Fig. 3 E, 3 H, Suppl. Figure 2A ), whereas siRNA-mediated knockdown of Bad or Bmf did not (Fig. 3 F, 3 G, 3 H, Suppl. Figure 2B, 2C ). Disruption of Noxa decreases hepatocyte apoptosis in Bcl-xL Mcl-1-, Bid-, Bim- and Puma-deficient mice and Mcl-1-deficient mice. We disrupted Noxa using CRISPR-Cas9 technology to generate tamoxifen-inducible Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bid −/− Bim −/− Puma −/− Noxa −/− mice (Fig. 4 A, Suppl. Figure 3A, 3B ). Disruption of Noxa significantly reduced the serum ALT levels and caspase 3/7 activity in tamoxifen-treated Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bid −/− Bim −/− Puma −/ mice (Fig. 4 B- 4 C). The number of TUNEL-positive hepatocytes was also significantly decreased by Noxa disruption (Fig. 4 D). Although some Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bid −/− Bim −/− Puma −/− Noxa −/− mice died 5 days after tamoxifen injection, Noxa disruption significantly prolonged their survival (Fig. 4 E). To discern the potential contribution of Noxa to hepatocyte apoptosis, particularly in the presence of Bid, Bim and Puma, we knocked out Noxa in Mcl-1 ΔHep/ΔHep mice (Fig. 5 A). Compared with Noxa+/+ mice, Noxa −/− mice showed no differences in serum ALT levels, serum caspase 3/7 activity or number of TUNEL-positive cells (Fig. 5 B- 5 D). Compared with Mcl-1 ΔHep/ΔHep mice, Mcl-1 ΔHep/ΔHep Noxa −/− mice exhibited significantly lower serum ALT levels and serum caspase 3/7 activity as well as fewer TUNEL-positive hepatocytes in liver sections (Fig. 5 B- 5 D). No BH3-only proteins other than Bid, Bim, Puma and Noxa contribute to hepatocyte apoptosis caused by the deletion of both Mcl-1 and Bcl-xL. To examine the role of the remaining BH3-only proteins in hepatocyte apoptosis caused by the deletion of both Mcl-1 and Bcl-xL, we generated a doxycycline-inducible Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bid −/− Bim −/− Puma −/− Noxa −/− hepatocyte cell line using immortalized primary mouse hepatocytes. Caspase 3/7 activity and LDH activities were significantly increased, and the relative cell viability was significantly decreased after incubation with doxycycline (Fig. 6 A, 6 B). Among the four remaining BH3-only proteins, Bad and Bmf were expressed in Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bid −/− Bim −/− Puma −/− Noxa −/− cells. After doxycycline treatment, Bad and Bmf mRNA expression levels increased (Fig. 6 C). However, siRNA-mediated knockdown of either of these genes did not affect caspase 3/7 activity, LDH activities or relative cell viability in doxycycline-treated Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bid −/− Bim −/− Puma −/− Noxa −/− cells (Fig. 6 D, 6 E). On the other hand, siRNA-mediated knockdown of both Bak and Bax completely abrogated the increases in caspase 3/7 activity and LDH activities and decreased cell viability (Fig. 6 F- 6 H). To explore other potential activators outside the realm of BH3-only proteins, we performed RNA-seq of a doxycycline-induced Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bid −/− Bim −/− Puma −/− Noxa −/− immortalized hepatocyte cell line and a doxycycline-induced Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bid −/− Bim −/− Puma −/− Noxa +/+ immortalized hepatocyte cell line (Fig. 3 A, 6 A). RNA-seq analysis revealed 977 genes in Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bid −/− Bim −/− Puma −/− Noxa −/− hepatocytes with FPKM values greater than twice those in Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bid −/− Bim −/− Puma −/− Noxa +/+ hepatocytes ( Suppl. Table 1 ). Among these genes, no Bcl-2 family proteins were detected, while Caspase6 , Caspase9 and Caspase12 , which are associated with apoptosis 11 , 12 , were detected. In the present study, we focused on caspase6 , which is known as an executor caspase, as well as caspase3/caspase7. SiRNA-mediated knockdown of caspase6 slightly but significantly increased relative cell viability in doxycycline-treated Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bid −/− Bim −/− Puma −/− Noxa −/− cells, while it did not affect caspase-3/7 activity ( Suppl. Figure 4A, 4B ). Similarly, siRNA-mediated knockdown of caspase6 slightly increased the relative viability of BNL.CL.2 cells, a murine hepatocyte cell line, treated with ABT-737, a Bcl-xL inhibitor that induces Bak/Bax-dependent hepatocyte apoptosis 13 without affecting caspase-3/7 activity ( Suppl. Figure 5A ). Discussion While the Bak/Bax-dependent mitochondrial pathway of apoptosis by Bcl-2 family proteins is precisely analyzed in vitro 8 , 14 , 15 , which BH3-only proteins among Bid, Bim, Puma, Noxa, Bad, Bik, Bmf and Hrk contribute to hepatocyte Bak/Bax activation in the healthy liver remains to be clarified. In the present study, we demonstrated that not only Bim and Bid but also Puma and Noxa exist in a functionally activated state under physiological conditions, collaboratively inducing Bak/Bax-dependent hepatocyte apoptosis when anti-apoptotic Bcl-2 family proteins in hepatocytes are deficient in vivo. Overall, hepatocyte integrity is maintained through the coordination of antiapoptotic bcl-2 family proteins with Bak/Bax activation mediated by Bid, Bim, Puma, and Noxa among the eight BH3-only proteins. We demonstrated that disruption of Puma attenuated hepatocyte apoptosis induced by the absence of Bcl-xL or Mcl-1 in vivo (Fig. 1 A- 1 F). This result suggested that Puma induces hepatocyte Bak/Bax activation, which is consistent with previous reports in vitro or in mouse embryonic fibroblasts (MEFs) 8 , 16 . Additionally, we showed that disruption of Noxa significantly reduced hepatocyte apoptosis induced by the absence of Mcl-1 (Fig. 5 A- 5 F). In Bid −/− Bim −/− Puma −/− mice, additional Noxa knockout mitigated hepatocyte apoptosis induced by the deletion of Bcl-xL and Mcl-1 (Fig. 4 A- 4 D). These results revealed that Noxa also plays a distinctive role in inducing hepatocyte apoptosis. Notably, there has been no previous in vivo study on the role of Puma and Noxa in hepatocyte Bak/Bax-dependent apoptosis in the healthy liver, and for the first time, we clarified their roles using an in vivo model. To explore additional activators beyond Bid, Bim, Puma and Noxa, we analyzed RNA-seq data, and we focused on caspase6 in the present study ( Suppl. Table 1 ). We showed that caspase6 is involved in hepatocyte apoptosis following Bak/Bax activation ( Suppl. Figure 4A, 4B, Suppl. Figure 5A ). It has been reported that caspase6 mediates a positive feedback loop to sustain the caspase cascade in hepatocytes via the AMPK-caspase6 pathway in nonalcoholic steatohepatitis 17 , although this feedback loop is not consistent with our findings because caspase6 knockdown had no effect on caspase-3/7 activity ( Suppl. Figure 4B, 5A ). Another report indicated that caspase-3, -6 and − 7 have nonredundant roles and that caspase6 operates downstream of caspase3, contributing partially to the induction of apoptosis 18 . The results of our study aligns with this finding. Further investigation is required to determine whether caspase6 partially regulates Bak/Bax-dependent hepatocyte apoptosis. In the present study, Bid −/− Bim −/− Puma −/− Noxa −/− mice still exhibited hepatocyte apoptosis after the knockout of Bcl-xL and Mcl-1 (Fig. 4 A- 4 D), and these mice exhibited markedly elevated serum ALT levels, increased caspase 3/7 activity and numerous TUNEL-positive hepatocytes on day 5 after tamoxifen injection (data not shown). However, the majority of these mice survived for 5 days (Fig. 4 E). To further confirm the mode of cell death in Bcl-xL- and Mcl-1-deficient hepatocytes in vivo, we generated Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bax iΔHep/iΔHep Bak −/− mice. Tamoxifen injection did not increase the serum ALT levels of these mice compared to those without tamoxifen injection ( Suppl. Figure 1A) , indicating that Bax/Bak deficiency completely blocked the hepatocyte apoptosis induced by Mcl-1/Bcl-xL deficiency. Notably, Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bax iΔHep/iΔHep Bak −/− mice without tamoxifen injection had higher serum ALT levels than Bcl-xL +/+ Mcl-1 +/+ Bax +/+ Bak −/− mice. This is most likely due to the leakage of Cre ERT 2 , as the expression levels of Bcl-xL and Bak were lower in Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bax iΔHep/iΔHep Bak −/− mice than in Bcl-xL +/+ Mcl-1 +/+ Bax +/+ Bak −/− mice even without tamoxifen injection ( Suppl. Figure 1B) . Moreover, all Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bax iΔHep/iΔHep Bak −/− mice survived for 6 days after tamoxifen injection, similar to control mice (Bcl-xL +/+ Mcl-1 +/+ Bax +/+ Bak −/− ) ( Suppl. Figure 1C ), while Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bid −/− Bim −/− Puma −/− Noxa −/− mice died by day 6 after tamoxifen injection (data not shown). These findings demonstrate that knocking out Bcl-xL and Mcl-1, even in the absence of the BH3-only proteins Bid, Bim, Puma and Noxa, still induces Bak/Bax-dependent hepatocyte apoptosis in vivo. A previous in vitro study suggested that Bak and Bax are automatically activated when both Bcl-xL and Mcl-1 are blocked in the absence of BH3-only proteins 8 , 19 . This mechanism could explain why Bid −/− Bim −/− Puma −/− Noxa −/− mice still exhibited hepatocyte apoptosis after the knockout of Bcl-xL and Mcl-1 in the present study. In conclusion, we elucidated the orchestrated regulation of Bcl-2 family proteins in hepatocyte apoptosis within a healthy liver, highlighting the roles of the BH3-only proteins Bid, Bim, Puma and Noxa. No other BH3-only proteins other than Bid, Bim, Puma and Noxa are involved in the Bak/Bax-dependent hepatocyte apoptosis induced by Bcl-xL/Mcl-1 deficiency. Understanding the orchestration of these Bcl-2 family proteins in hepatocytes provides insights into hepatocyte homeostasis and pathogenesis related to hepatocyte apoptosis. Materials and methods Mice Puma knockout mice ( Puma −/− ; Puma −/− mice ) and Bim knockout mice ( Bim −/− ; Bim −/− mice) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Bid knockout mice ( Bid −/− ; Bid −/− mice) were generated as described previously 20 . Bak/Bax double knockout mice ( Bax flox/flox Alb-Cre Bak −/− ; Bax ΔHep/ΔHep Bak −/− mice) were generated as described previously 5 . Hepatocyte-specific Bcl-xL knockout mice ( Bcl-xL flox/flox Alb-Cre ; Bcl-xL ΔHep/ΔHep mice) 6 and hepatocyte-specific Mcl-1 knockout mice (Mcl-1 flox/flox Alb-Cre ; Mcl-1 ΔHep/ΔHep mice) 10 were crossed with Puma −/− mice to produce Bcl-xL ΔHep/ΔHep Puma −/− mice and Mcl-1 ΔHep/ΔHep Puma −/− mice, respectively. Mated Bcl-xL flox/flox Alb-Cre Puma +/− and Bcl-xL flox/flox Puma +/− , Mcl-1 flox/flox Alb-Cre Puma +/− and Mcl-1 flox/flox Puma +/ offspring were analyzed at 6 to 8 weeks of age. We further crossed Bcl-xL ΔHep/ΔHep mice, Mcl-1 ΔHep/ΔHep mice, Bid −/− mice, Bim −/− mice and Puma −/− mice to generate Bcl-xL ΔHep/ΔHep Mcl-1 ΔHep/+ Bid −/− Bim −/− Puma −/− mice. The offspring of Bcl-xL flox/flox Mcl-1 flox/+ Alb-Cre Bid −/− Bim −/− (or Bim +/− ) Puma +/− mice and Bcl-xL flox/flox Mcl-1 flox/flox Bid −/− Bim −/− (or Bim +/− ) Puma +/− mice were analyzed at 6 to 8 weeks of age. Of note, for this mating, we used Bim −/− male and Bim +/− female mice because Bim −/− female mice are infertile. We also crossed Bcl-xL ΔHep/ΔHep mice, Mcl-1 ΔHep/ΔHep mice with Bax ΔHep/ΔHep Bak −/− mice to generate Bcl-xL ΔHep/ΔHep Mcl-1 ΔHep/+ Bax ΔHep/ΔHep Bak −/− mice. The offspring of Bcl-xL flox/flox Mcl-1 flox/+ Bax flox/flox Alb-Cre Bak +/− mice and Bcl-xL flox/flox Mcl-1 flox/flox Bax flox/flox Bak +/− mice were analyzed at 6 to 8 weeks of age. Tamoxifen-inducible hepatocyte-specific Cre mice ( Albumin-Cre-ERT2 ) were kindly provided by Professor Pierre Chambon 21 , and we generated tamoxifen-inducible hepatocyte-specific Bcl-xL and Mcl-1 knockout mice ( Bcl-xL flox/flox Mcl-1 flox/flox Alb- Cre ERT 2 ; Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep mice) and Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bid −/− Bim −/− Puma −/− mice by crossing them. We also generated Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bax iΔHep/iΔHep Bak −/− mice by crossing them with Bax ΔHep/ΔHep Bak −/− mice. The mice were injected intraperitoneally with 1 mg of tamoxifen (Sigma‒Aldrich, St. Louis, MO, USA) at the indicated times. The animals were housed in cages under specific pathogen-free conditions with free access to water and standard mouse chow. Generation of Noxa knockout mice Fertilized eggs from Bcl-xL flox/flox Mcl-1 flox/flox Alb-Cre ERT 2 Bid −/− Bim +/− Puma −/− mice were produced and used for in vitro fertilization. Subsequently, Noxa knockout guide RNA (gRNA) and Cas9 proteins were introduced into these fertilized eggs through electroporation. gRNA was designed using a software tool ( http://crispor.tefor.net/and https://crispr.dbcls.jp/ ) to predict unique target sites throughout the mouse genome. Cas9 proteins were obtained from Alt-R® S.p.Cas9 Nucleases 3NLS (Integrated DNA Technologies, Inc., USA). Deletion of the Noxa sequence was confirmed by genotyping DNA isolated from the resulting offspring ( Suppl. Figure 3A, 3B ). We generated Bcl-xL iΔHep/iΔHep Mcl-1 iΔHep/iΔHep Bid −/− Bim −/− Puma −/− Noxa −/− mice by crossing Bcl-xL flox/flox Mcl-1 flox/flox Alb-Cre ERT 2 Bid −/− Bim −/− (or Bim +/− ) Puma −/− Noxa +/− mice with Bcl-xL flox/flox Mcl-1 flox/flox Bid −/− Bim +/− (or Bim −/− ) Puma −/− Noxa +/− mice and analyzed their offspring. Quantitative real-time RT‒PCR analysis Total RNA was extracted from cells and liver tissues by using an RNeasy Kit (QIAGEN, Hilden, Germany) and reverse transcribed using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan). Real-time RT‒PCR was performed by using TaqMan gene expression assays with an HT7900 Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The following TaqMan gene expression primers were used: Noxa (Mm00451763_m1), Bad (Mm00432042_m1), Bmf (Mm00506773_m1), Bik (Mm00476123_m1), Hrk (Mm01208086_m1), Bax (Mm00432051_m1), Bak (Mm00432045_m1), Caspase6 (Mm01321726_g1) and Actb (Mm02619580_g1). Gene expression levels were normalized to those of Actb . Western blotting analysis Whole-cell extracts and liver tissues were lysed in lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, phosphate-buffered saline, pH 7.4, 0.1% sodium dodecyl sulfate, 1× proteinase inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and 1× phosphatase inhibitor cocktail (Nacalai Tesque). The supernatant was collected by centrifugation (13,500 × g for 30 min at 4°C), and the protein concentrations were determined by a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Protein samples of equal concentration were separated on sodium dodecyl sulfate polyacrylamide gels and transferred onto polyvinylidene fluoride membranes. For immunodetection, the following antibodies were used: an anti-Bid antibody (#2003), anti-Bim antibody (#2933), anti-Puma antibody (#24633S), and anti-Bad antibody (#9292) purchased from Cell Signaling Technology (Beverly, MA, USA); an anti-Mcl-1 antibody (600-401-394) purchased from Rockland Immunochemicals (Limerick, PA, USA); an anti-Bcl-xL antibody (sc-634) purchased from Santa Cruz Biotechnology (Dallas, TX, USA); an anti-Noxa antibody (ab23563) and anti-Bmf antibody (ab9655) purchased from Abcam (Cambridge, MA, USA); and an anti-β-actin antibody (A5316) purchased from Sigma‒Aldrich. Small interfering RNA (siRNA)-mediated knockdown Immortalized cells were transfected with 10 nM siRNA by Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer’s protocol. The cells were treated with 0.3 µM doxycycline (Wako Pure Chemical Industries, Osaka, Japan) for 24 hours and then analyzed 72 hours after transfection. The following siRNAs were used: siRNA against Noxa (s81669), siRNA against Bad (s233500), siRNA against Bmf (s101192), siRNA against Bak (s62860), siRNA against Bax (s62874), and siRNA against Caspase6 (s63387). The appropriate negative controls were purchased from Thermo Fisher Scientific. Hepatic function, caspase activity, and hepatocyte death analyses Serum alanine transaminase (ALT) levels were measured by using a DRI-CHEM NX700iV (FUJIFILM, Tokyo, Japan). Serum caspase 3/7 activity was measured with a luminescent substrate assay for caspase-3 and caspase-7 (Caspase-Glo Assay, Promega, Madison, WI, USA) according to the manufacturer’s protocol and is shown as relative values. Liver sections were stained with hematoxylin and eosin (HE). To detect apoptotic hepatocytes, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was performed with an ApopTag Kit (Millipore, Moldheim, France) according to the manufacturer’s protocol. TUNEL-positive cells were counted in four fields per liver section, and the average number of TUNEL-positive cells was determined. In vitro cell death assay Cell viability was measured by a water-soluble tetrazolium salt (WST) assay (Nakalai Tesque), and the results are shown as relative values. Caspase3/7 activity was measured with a luminescent substrate assay for caspase-3 and caspase-7 (Promega) according to the manufacturer’s protocol and is shown as relative values. The LDH activities of cultured cells was measured using a Cytotoxicity LDH Assay Kit-WST (Dojindo Laboratories, Kumamoto, Japan), and the results are shown as relative values. ABT-737 was purchased from Selleck Chemicals (Houston, TX, USA). An IncuCyte SX1 live-cell analysis system (Sartorius Japan, Tokyo, Japan) was used to analyze the apoptosis of immortalized cells cultured with doxycycline. Twenty-four hours after siRNA-mediated knockdown, Annexin V Green Reagent for Apoptosis (Sartorius, Japan) was added to the culture medium of the immortalized cells at the same time as doxycycline. Images of Annexin V-positive areas at different time points were obtained and the fluorescence intensity was normalized to that of the Annexin V-positive areas at 0 hours. All of the images were analyzed by using IncuCyte 2021A software (Sartorius Japan). Primary hepatocyte culture, isolation and immortalization Mouse primary hepatocytes were isolated using the two-step collagenase-pronase liver perfusion method in the same manner as previously reported 13 . Isolated hepatocytes were cultured in William’s Eagle medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FCS), 2 mM L-glutamine (Thermo Fisher Scientific), 100 nM insulin (Sigma‒Aldrich) and 100 nM dexamethasone (Sigma‒Aldrich). To immortalize mouse primary hepatocytes, they were transfected with a lentiviral vector expressing simian virus 40 large T antigen (SV40T) purchased from Addgene (Watertown, MA, USA, plasmid #22298). BNL CL.2 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma‒Aldrich) in an incubator with 5% CO 2 at 37°C. The medium contained 10% fetal bovine serum and 1% antibiotics (Anti-Anti; Thermo Fisher). Generation of immortalized cells with doxycycline-dependent Cre/LoxP recombination The plasmid (pLenti-Cre-IRES-PuroR, Addgene #30205) was amplified by PCR and electrophoresed on a 0.8% gel. The 1029 bp band was eluted from the gel and used as an insert. The vector (pLenti-iCas9-neo, Addgene #85400) was cleaved by two restriction enzymes, XhoI and BsmBI (Bio-Rad, Hercules, CA, USA), and electrophoresed on a 0.8% gel. The 9959 bp band was eluted and used as a backbone. Using an In-Fusion cloning kit (Takara Bio Inc., Shiga, Japan) with these inserts and backbones, a plasmid with doxycycline-inducible Cre and neomycin resistance gene constructs (pLenti-iCre-neo) was prepared. The obtained plasmids were transfected into HEK293 cells for lentivirus packaging. The viral supernatant was used to transfect the immortalized primary hepatocytes. These cells were cultured with 700 µg/ml neomycin (Thermo Fisher Scientific) for 1 week, and pLenti-iCre-Neo-transfected immortalized cells were selected. Transfected immortalized cells were then subjected to the limiting dilution method to generate a monoclonal stable cell line. Statistical analysis Statistical analysis was performed using GraphPad Prism 9.4.0 (GraphPad, La Jolla, CA, USA). Two-tailed unpaired t tests were used to compare the differences between two groups. One-way ANOVA with Sidak’s multiple comparisons test or Dunnett's multiple comparisons test was performed to compare multiple groups. The Kaplan‒Meier method was used for survival analysis. A P value < 0.05 was considered to indicate statistical significance. All of the data are expressed as the means ± SDs. All of the in vitro experiments were repeated at least three times unless otherwise indicated. Declarations Funding Supported by Grants-in-Aid for Scientific Research from the Japan Agency for Medical Research and Development (JP23fk0210121 and JP23fk0310512 to T. Takehara) and Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, Japan (JP21H02903 to T. Takehara and JP23H02894 to H.H.). Data availability All data will be made available immediately after publication on request. Acknowledgments We would like to express our gratitude to Dr. Yuko Kotani and their colleagues from The Institute of Experimental Animal Sciences, Faculty of Medicine, Osaka University, for generating Noxa knockout mice using CRISPR-Cas9 technology and Mayumi Honma at Osaka University Graduate School of Medicine for performing the outstanding immunohistochemistry experiments. Authors and Affiliations Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan Shinnosuke Kudo § , Hayato Hikita, Yoshinobu Saito § , Kazuhiro Murai, Takahiro Kodama, Tomohide Tatsumi, Tetsuo Takehara § These authors contributed equally: Shinnosuke Kudo, Yoshinobu Saito Author Contributions S.K. and Y.S. conceived and designed the experiments; generated, analyzed, and interpreted the data; and drafted the manuscript. K.M., T.K. and T. Tatsumi provided conceptual input. H.H. and T. Takehara conceived and oversaw the study, designed the experiments, and drafted and edited the manuscript. Competing interests These authors declare no competing interests. Ethics approval All of the mice were treated humanely, and all of the animal experiments were approved by the Animal Care and Use Committee of Osaka University Medical School (Osaka, Japan) and the Animal Care Committee of Central Institute for Experimental Animals. Funding Supported by Grants-in-Aid for Scientific Research from the Japan Agency for Medical Research and Development (JP23fk0210121 and JP23fk0310512 to T. Takehara) and Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, Japan (JP21H02903 to T. Takehara and JP23H02894 to H.H.). References Czabotar PE, Garcia-Saez AJ. Mechanisms of BCL-2 family proteins in mitochondrial apoptosis. Nature Reviews Molecular Cell Biology 2023, 24(10): 732–748. Singh R, Letai A, Sarosiek K. Regulation of apoptosis in health and disease: the balancing act of BCL-2 family proteins. Nat Rev Mol Cell Biol 2019, 20(3): 175–193. Kaloni D, Diepstraten ST, Strasser A, Kelly GL. BCL-2 protein family: attractive targets for cancer therapy. Apoptosis 2023, 28(1–2): 20–38. Glab JA, Mbogo GW, Puthalakath H. BH3-Only Proteins in Health and Disease. International Review of Cell and Molecular Biology, Vol 328 2017, 328: 163–196. Hikita H, Takehara T, Kodama T, Shimizu S, Hosui A, Miyagi T, et al. BH3-Only Protein Bid Participates in the Bcl-2 Network in Healthy Liver Cells. Hepatology 2009, 50(6): 1972–1980. Takehara T, Tatsumi T, Suzuki T, Rucker EB, 3rd, Hennighausen L, Jinushi M, et al. Hepatocyte-specific disruption of Bcl-xL leads to continuous hepatocyte apoptosis and liver fibrotic responses. Gastroenterology 2004, 127(4): 1189–1197. Hikita H, Kodama T, Shimizu S, Li W, Shigekawa M, Tanaka S, et al. Bak deficiency inhibits liver carcinogenesis: A causal link between apoptosis and carcinogenesis. Journal of Hepatology 2012, 57(1): 92–100. Chen HC, Kanai M, Inoue-Yamauchi A, Tu HC, Huang YF, Ren DC, et al. An interconnected hierarchical model of cell death regulation by the BCL-2 family. Nature Cell Biology 2015, 17(10): 1270-+. Kodama T, Hikita H, Kawaguchi T, Saito Y, Tanaka S, Shigekawa M, et al. The Bcl-2 homology domain 3 (BH3)-only proteins Bim and bid are functionally active and restrained by anti-apoptotic Bcl-2 family proteins in healthy liver. J Biol Chem 2013, 288(42): 30009–30018. Hikita H, Takehara T, Shimizu S, Kodama T, Li W, Miyagi T, et al. Mcl-1 and Bcl-xL Cooperatively Maintain Integrity of Hepatocytes in Developing and Adult Murine Liver. Hepatology 2009, 50(4): 1217–1226. Kesavardhana S, Malireddi RKS, Kanneganti TD. Caspases in Cell Death, Inflammation, and Pyroptosis. Annu Rev Immunol 2020, 38: 567–595. Nakagawa T, Zhu H, Morishima N, Li E, Xu J, Yankner BA, et al. Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature 2000, 403(6765): 98–103. Saito Y, Hikita H, Nozaki Y, Kai Y, Makino Y, Nakabori T, et al. DNase II activated by the mitochondrial apoptotic pathway regulates RIP1-dependent non-apoptotic hepatocyte death via the TLR9/IFN-beta signaling pathway. Cell death and differentiation 2019, 26(3): 470–486. King L, Rodriguez-Enriquez R, Pedley R, Mellor C, Wang P, Zindy E, et al. Apoptotic priming is defined by the dynamic exchange of Bcl-2 proteins between mitochondria and cytosol. Cell death and differentiation 2022, 29: 2262–2274. Yamazaki T, Galluzzi L. BAX and BAK dynamics control mitochondrial DNA release during apoptosis. Cell death and differentiation 2022, 29: 1296–1298. Kim H, Tu HC, Ren DR, Takeuchi O, Jeffers JR, Zambetti GP, et al. Stepwise Activation of BAX and BAK by tBID, BIM, and PUMA Initiates Mitochondrial Apoptosis. Molecular Cell 2009, 36(3): 487–499. Zhao P, Sun X, Chaggan C, Liao Z, In Wong K, He F, et al. An AMPK-caspase-6 axis controls liver damage in nonalcoholic steatohepatitis. Science 2020, 367(6478): 652–660. Slee EA, Adrain C, Martin SJ. Executioner caspase-3, -6, and – 7 perform distinct, non-redundant roles during the demolition phase of apoptosis. J Biol Chem 2001, 276(10): 7320–7326. Huang K, O'Neill KL, Li J, Zhou W, Han N, Pang XM, et al. BH3-only proteins target BCL-xL/MCL-1, not BAX/BAK, to initiate apoptosis. Cell Research 2019, 29(11): 942–952. Yin XM, Wang K, Gross A, Zhao YG, Zinkel S, Klocke B, et al. Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis. Nature 1999, 400(6747): 886–891. Schuler M, Dierich A, Chambon P, Metzger D. Efficient temporally controlled targeted somatic mutagenesis in hepatocytes of the mouse. Genesis 2004, 39(3): 167–172. Tables Tables 1 to 2 are available in the Supplementary Files section Additional Declarations There is no duality of interest Supplementary Files Suppl.Figure1.tif Suppl.Figure2.tif Suppl.Figure3.tif Suppl.Figure4.tif Suppl.Figure5.tif Suppl.Table1.xlsx Suppl.FigureLegend.docx originalwesternblots.pdf Cite Share Download PDF Status: Published Journal Publication published 24 Feb, 2025 Read the published version in Cell Death & Differentiation → Version 1 posted Editorial decision: revise 01 Aug, 2024 Review # 3 received at journal 31 Jul, 2024 Review # 1 received at journal 18 Jul, 2024 Reviewer # 3 agreed at journal 17 Jul, 2024 Review # 2 received at journal 16 Jul, 2024 Reviewer # 2 agreed at journal 15 Jul, 2024 Reviewer # 1 agreed at journal 15 Jul, 2024 Reviewers invited by journal 08 Jul, 2024 Submission checks completed at journal 08 Jul, 2024 Editor assigned by journal 08 Jul, 2024 First submitted to journal 08 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4704982","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":324166677,"identity":"bc9a4dd4-9959-4eee-9f3d-3688c21fa829","order_by":0,"name":"Tetsuo Takehara","email":"data:image/png;base64,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","orcid":"","institution":"Osaka University Graduate School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Tetsuo","middleName":"","lastName":"Takehara","suffix":""},{"id":324166678,"identity":"dedb90a5-663b-419e-96cf-d318db165487","order_by":1,"name":"Shinnosuke Kudo","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Shinnosuke","middleName":"","lastName":"Kudo","suffix":""},{"id":324166679,"identity":"8a25715f-ae3f-4ded-8b8f-f1df591a6a3c","order_by":2,"name":"Hayato Hikita","email":"","orcid":"","institution":"Osaka University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hayato","middleName":"","lastName":"Hikita","suffix":""},{"id":324166680,"identity":"eae72e2a-9186-45d9-a1bf-1032438d5472","order_by":3,"name":"Yoshinobu Saito","email":"","orcid":"","institution":"Osaka University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yoshinobu","middleName":"","lastName":"Saito","suffix":""},{"id":324166681,"identity":"bee1279e-1dd2-435a-a0a6-59f5b0402651","order_by":4,"name":"Kazuhiro Murai","email":"","orcid":"","institution":"Osaka University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kazuhiro","middleName":"","lastName":"Murai","suffix":""},{"id":324166682,"identity":"93852480-47cb-46c7-bf44-0541dc9493f0","order_by":5,"name":"Takahiro Kodama","email":"","orcid":"https://orcid.org/0000-0002-6250-1324","institution":"Osaka University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Takahiro","middleName":"","lastName":"Kodama","suffix":""},{"id":324166683,"identity":"0a102cab-770d-4f23-8dff-92114cbb8aa9","order_by":6,"name":"Tomohide Tatsumi","email":"","orcid":"","institution":"Osaka University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Tomohide","middleName":"","lastName":"Tatsumi","suffix":""}],"badges":[],"createdAt":"2024-07-08 10:51:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4704982/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4704982/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41418-025-01458-y","type":"published","date":"2025-02-24T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61507524,"identity":"18a06d09-1204-4390-b307-7cea02e15826","added_by":"auto","created_at":"2024-07-31 14:05:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":849056,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDisruption of Puma suppresses hepatocyte apoptosis in mice with hepatocyte-specific knockout of Bcl-xL or Mcl-1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe generated \u003cem\u003eBcl-xL\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Alb-Cre Puma\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice, \u003cem\u003eMcl-1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Alb-Cre Puma\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice and control mice. These mice were analyzed at 6 to 8 weeks of age; n≥4 per group. (\u003cstrong\u003eA, B\u003c/strong\u003e) Western blotting analysis of liver lysates. (\u003cstrong\u003eC, D\u003c/strong\u003e) Serum ALT levels and caspase3/7 activities. (\u003cstrong\u003eE, F\u003c/strong\u003e) Representative images of H\u0026amp;E staining and TUNEL staining, and their quantitative results. Data is represented as mean ± SD. Data was analyzed by One-way ANOVA with Sidak's multiple comparisons test (\u003cstrong\u003eC-F\u003c/strong\u003e). Scale bar (\u003cstrong\u003eE, F\u003c/strong\u003e): 100 μm\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4704982/v1/406da34aa73859e04b9e6a04.png"},{"id":61508184,"identity":"fc755df7-a8a2-4193-b506-3550cc0f17cf","added_by":"auto","created_at":"2024-07-31 14:13:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":618280,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDisruption of Bid, Bim and Puma cannot fully suppress hepatocyte apoptosis induced by the deletion of Bcl-xL and Mcl-1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA-D\u003c/strong\u003e) We generated \u003cem\u003eBcl-xL\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Mcl-1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Alb-Cre Bid\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eBim\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003e\u003cem\u003ePuma\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice, \u003cem\u003eBcl-xL\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Mcl-1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Alb-Cre Bid\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eBim\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003e\u003cem\u003ePuma\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice, and control mice. We also generated \u003cem\u003eBcl-xL\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Mcl-1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Bax\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Alb-Cre Bak\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e-\u003c/sup\u003e mice. These mice were analyzed at 6 to 8 weeks of age; n=6 per group. (\u003cstrong\u003eA\u003c/strong\u003e) Western blotting analysis of liver lysates. (\u003cstrong\u003eB, C\u003c/strong\u003e) Serum ALT levels and Caspase3/7 activities. (\u003cstrong\u003eD\u003c/strong\u003e) Representative images of H\u0026amp;E staining and TUNEL staining, and their quantitative results. (\u003cstrong\u003eE-H\u003c/strong\u003e) We generated \u003cem\u003eBcl-xL\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Mcl-1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eAlb-Cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT2\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eBid\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Bim\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Puma\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice, and control mice. These mice were injected intraperitoneally with 1mg of tamoxifen for 3 consecutive days and analyzed 24 hours after last tamoxifen injection. (\u003cstrong\u003eE\u003c/strong\u003e) Western blotting analysis of liver lysates after tamoxifen injection. (\u003cstrong\u003eF, G\u003c/strong\u003e) Serum ALT levels and caspase3/7 activities. (\u003cstrong\u003eH\u003c/strong\u003e) Representative images of H\u0026amp;E staining and TUNEL staining, and their quantitative results. \u0026nbsp;Data is represented as mean ± SD. Data was analyzed by One-way ANOVA with Sidak's multiple comparisons test (\u003cstrong\u003eB-D\u003c/strong\u003e) or two-tailed unpaired\u0026nbsp;\u003cem\u003et\u003c/em\u003e-test (\u003cstrong\u003eF-H\u003c/strong\u003e). Scale bar (\u003cstrong\u003eD, H\u003c/strong\u003e): 100 μm\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4704982/v1/adff8bef3df83b9d0c26adf7.png"},{"id":61508186,"identity":"e868f78f-5680-44e8-b99e-b0e9443a8849","added_by":"auto","created_at":"2024-07-31 14:13:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":151771,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNoxa knockdown suppresses apoptosis in Bcl-xL-, Mcl-1-, Bid-, Bim- and Puma-deficient hepatocytes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA, B\u003c/strong\u003e) Immortalized \u003cem\u003eBcl-xL\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Mcl-1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eBid\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Bim\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Puma\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emouse primary hepatocytes with doxycycline-inducible Cre recombinase were incubated with 0.3 µM doxycycline for 48 hours. (\u003cstrong\u003eA\u003c/strong\u003e) Western blot analysis. (\u003cstrong\u003eB\u003c/strong\u003e) Caspase-3/7 activity and LDH activity in culture supernatants, cell viability assessed with a WST assay. (\u003cstrong\u003eC\u003c/strong\u003e) Relative mRNA expression levels of BH3-only proteins in the immortalized cell. (\u003cstrong\u003eD\u003c/strong\u003e) Western blot analysis of Noxa, Bad and Bmf expression levels after doxycycline incubation. (\u003cstrong\u003eE-G\u003c/strong\u003e) 24 hours after transfection with \u003cem\u003eNoxa, Bad, Bmf\u003c/em\u003e siRNA or control siRNA, immortalized \u003cem\u003eBcl-xL\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Mcl-1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eBid\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Bim\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Puma\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emouse primary hepatocytes were treated with 0.3 μM doxycycline for 48 hours. Caspase-3/7 activity and LDH activity in the culture supernatant, cell viability assessed with a WST assay after (\u003cstrong\u003eE\u003c/strong\u003e)\u003cem\u003e Noxa\u003c/em\u003e, (\u003cstrong\u003eF\u003c/strong\u003e) \u003cem\u003eBad\u003c/em\u003e, and (\u003cstrong\u003eG\u003c/strong\u003e) \u003cem\u003eBmf \u003c/em\u003eknockdown. (\u003cstrong\u003eH\u003c/strong\u003e) Annexin V positive cell areas after transfection with \u003cem\u003eNoxa, Bad, Bmf\u003c/em\u003e siRNA or control siRNA and doxycycline incubation. Data is represented as mean ± SD. Data was analyzed by two-tailed unpaired\u0026nbsp;\u003cem\u003et\u003c/em\u003e-test (\u003cstrong\u003eB\u003c/strong\u003e), One-way ANOVA with Sidak's multiple comparisons test (\u003cstrong\u003eE-G\u003c/strong\u003e) or One-way ANOVA with Dunnett's multiple comparisons test (\u003cstrong\u003eH\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4704982/v1/2c53e25bb68ed8923612c04b.png"},{"id":61507528,"identity":"45c7fddb-6d77-4c84-a00e-8dd9673c683e","added_by":"auto","created_at":"2024-07-31 14:05:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":418766,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional disruption of Noxa significantly decreases hepatocyte apoptosis in Bcl-xL Mcl-1-, Bid-, Bim- and Puma-deficient\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe generated \u003cem\u003eBcl-xL\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Mcl-1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eBid\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Bim\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Puma\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Noxa\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Alb-Cre ERT2\u003c/em\u003e mice by using CRISPR/Cas9 technology. These mice were injected intraperitoneally with 1mg of tamoxifen and sacrificed after 12 hours; n=6 per group.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Expression of Mcl-1, Bcl-xL, Bid, Bim, Puma, Noxa and β-actin proteins assessed by western blotting analysis of liver lysates after tamoxifen injection. (\u003cstrong\u003eB, C\u003c/strong\u003e) Serum ALT levels and Caspase3/7 activities. (\u003cstrong\u003eD\u003c/strong\u003e) Representative images of H\u0026amp;E staining and TUNEL staining. (\u003cstrong\u003eE\u003c/strong\u003e) Probability of survival after consecutive tamoxifen injection (n=4-5). Data is represented as mean ± SD. Data was analyzed by One-way ANOVA with Sidak's multiple comparisons test (\u003cstrong\u003eB-D\u003c/strong\u003e) or log-rank test (\u003cstrong\u003eE\u003c/strong\u003e). Scale bar (\u003cstrong\u003eD\u003c/strong\u003e): 100 μm\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4704982/v1/8a669fca1dc70ea4216a89c1.png"},{"id":61508717,"identity":"cab311a4-404b-4f98-a72f-916d7bc7184a","added_by":"auto","created_at":"2024-07-31 14:21:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":378548,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDisruption of Noxa suppresses hepatocyte apoptosis in hepatocyte-specific Mcl-1 knockout mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe generated \u003cem\u003eMcl-1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Alb-Cre Noxa\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice and control mice. These mice were analyzed at 6 to 8 weeks of age; n=6 per group.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Expression of Mcl-1, Noxa, Bcl-xL, Bid, Bim, Puma and β-actin proteins assessed by western blot analysis. (\u003cstrong\u003eB, C\u003c/strong\u003e) Serum ALT levels and Caspase3/7 activities. (\u003cstrong\u003eD\u003c/strong\u003e) Representative images of H\u0026amp;E staining and TUNEL staining, and their quantitative results. Data is represented as mean ± SD. Data was analyzed by One-way ANOVA with Sidak's multiple comparisons test (\u003cstrong\u003eB-D\u003c/strong\u003e). Scale bar (\u003cstrong\u003eD\u003c/strong\u003e): 100 μm\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4704982/v1/e48e23ae1b97fa7480621f88.png"},{"id":61507535,"identity":"b76ec0ff-8be3-4cfe-b658-92091fca9db1","added_by":"auto","created_at":"2024-07-31 14:05:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":152754,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNo BH3-only proteins other than Bid, Bim, Puma and contribute to hepatocyte apoptosis caused by the deletion of both Mcl-1 and Bcl-xL.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA-C\u003c/strong\u003e) Immortalized \u003cem\u003eBcl-xL\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Mcl-1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eBid\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Bim\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Puma\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Noxa\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emouse primary hepatocytes with doxycycline-inducible Cre recombinase were incubated with 0.3 µM doxycycline for 48 hours. (\u003cstrong\u003eA\u003c/strong\u003e) Western Blot analysis. (\u003cstrong\u003eB\u003c/strong\u003e) Caspase-3/7 activity and LDH activity in culture supernatants, cell viability assessed with a WST assay. (\u003cstrong\u003eC\u003c/strong\u003e) Relative mRNA expression levels of \u003cem\u003eBad\u003c/em\u003e and \u003cem\u003eBmf\u003c/em\u003e after doxycycline incubation. (\u003cstrong\u003eD, E\u003c/strong\u003e) Immortalized \u003cem\u003eBcl-xL\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Mcl-1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eBid\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Bim\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Puma\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Noxa\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emouse primary hepatocytes were treated with 0.3 μM doxycycline for 48 hours after transfection of\u003cem\u003e Bad\u003c/em\u003e,\u003cem\u003e Bmf\u003c/em\u003e siRNA or control siRNA. Caspase-3/7 activity and LDH activity in culture supernatants, WST assay after (\u003cstrong\u003eD\u003c/strong\u003e) \u003cem\u003eBad\u003c/em\u003e and (\u003cstrong\u003eE\u003c/strong\u003e) \u003cem\u003eBmf\u003c/em\u003e knockdown. (\u003cstrong\u003eF-H\u003c/strong\u003e) Immortalized \u003cem\u003eBcl-xL\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Mcl-1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eBid\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Bim\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Puma\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e Noxa\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emouse primary hepatocytes were treated with 0.3 μM doxycycline for 48 hours after transfection of both\u003cem\u003e Bak\u003c/em\u003e and\u003cem\u003e Bax\u003c/em\u003e siRNA or control siRNA. (\u003cstrong\u003eF, G\u003c/strong\u003e) Relative mRNA expression levels of (\u003cstrong\u003eF\u003c/strong\u003e) \u003cem\u003eBak\u003c/em\u003e and (\u003cstrong\u003eG\u003c/strong\u003e) \u003cem\u003eBax\u003c/em\u003e after siRNA transfection. (\u003cstrong\u003eH\u003c/strong\u003e) Caspase-3/7 activity and LDH activity in culture supernatants, \u0026nbsp;WST assay after Bak and Bax knockdown. Data is represented as mean ± SD. Data was analyzed by two-tailed unpaired\u0026nbsp;\u003cem\u003et\u003c/em\u003e-test (\u003cstrong\u003eB, C, F, G\u003c/strong\u003e) or One-way ANOVA with Sidak's multiple comparisons test (\u003cstrong\u003eD, E, H\u003c/strong\u003e)\u003c/p\u003e","description":"","filename":"OnlineFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4704982/v1/737e9083f287c944e76d56d9.png"},{"id":77106109,"identity":"36050769-7b49-4b7d-9003-00a91fddc543","added_by":"auto","created_at":"2025-02-25 08:10:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5117877,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4704982/v1/bba7be67-24c8-4b9c-8723-2be3f2320ec5.pdf"},{"id":61507516,"identity":"6ea4731c-7dd7-4c77-ab78-91bc1276f84c","added_by":"auto","created_at":"2024-07-31 14:05:21","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":701014,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.Figure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4704982/v1/49f7feec7d7cba20cd86e8d5.tif"},{"id":61507518,"identity":"49bde2b4-77d0-4c88-bff4-fd5b41d029e0","added_by":"auto","created_at":"2024-07-31 14:05:21","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":540668,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.Figure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-4704982/v1/fabc4de6eb0b901f35651ea5.tif"},{"id":61507525,"identity":"8d4328dc-30ae-4ff2-a6e2-4d2d700787c4","added_by":"auto","created_at":"2024-07-31 14:05:21","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":735494,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.Figure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-4704982/v1/b7f9ad966e385f8dd0c0f547.tif"},{"id":61507529,"identity":"f3f87328-977f-497c-8cd5-b37d0d89f292","added_by":"auto","created_at":"2024-07-31 14:05:21","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":587994,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.Figure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-4704982/v1/c3e6a56e9e972a7ee736e508.tif"},{"id":61508188,"identity":"89ec710e-a1d3-4390-8c64-8164e2af79d2","added_by":"auto","created_at":"2024-07-31 14:13:21","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":566728,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.Figure5.tif","url":"https://assets-eu.researchsquare.com/files/rs-4704982/v1/198a63b15172ac9eb29eebda.tif"},{"id":61507532,"identity":"6b76e8b2-d471-44c8-bbb4-d33b4b774b8f","added_by":"auto","created_at":"2024-07-31 14:05:22","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":8475546,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4704982/v1/5bf744e5a94ebb81c51cf487.xlsx"},{"id":61508187,"identity":"433be670-9cdb-4db3-9785-e39529aae0f1","added_by":"auto","created_at":"2024-07-31 14:13:21","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":24370,"visible":true,"origin":"","legend":"","description":"","filename":"Suppl.FigureLegend.docx","url":"https://assets-eu.researchsquare.com/files/rs-4704982/v1/5a9cfcb102b8796fb34c9fcd.docx"},{"id":61507534,"identity":"0b7c698c-f36a-4fa1-b1ec-d5dc1ca1761c","added_by":"auto","created_at":"2024-07-31 14:05:23","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":629628,"visible":true,"origin":"","legend":"","description":"","filename":"originalwesternblots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4704982/v1/87c76df7a5d8cc095b9c0d26.pdf"}],"financialInterests":"There is no duality of interest","formattedTitle":"Collaborative orchestration of BH3-only proteins governs Bak/Bax-dependent hepatocyte apoptosis under antiapoptotic protein-deficiency in mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe mitochondrial pathway of hepatocyte apoptosis is orchestrated by members of B-cell lymphoma-2 (Bcl-2) family proteins, which include the anti-apoptotic proteins Bcl-2, B-cell lymphoma-extra large (Bcl-xL), myeloid cell leukemia-1 (Mcl-1), B-cell lymphoma-w (Bcl-w), and Bcl-2-related protein A1 (Bcl2A1), as well as the pro-apoptotic proteins Bcl-2-antagonist/killer (Bak) and Bcl-2-associated X protein (Bax)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Pro-apoptotic proteins act as effector molecules in this apoptotic cascade\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Upon activation, they form pores in the mitochondrial outer membrane, facilitating the release of cytochrome c. This leads to the activation of the caspase cascade and finally leads to apoptosis\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Anti-apoptotic Bcl-2 family members, including Bcl-xL and Mcl-1, inhibit the mitochondrial pathway of apoptosis by neutralizing Bak/Bax activity. Bcl-2 homology domain 3 (BH3)-only proteins, a group of eight members, Bid, Bim, Puma, Noxa, Bad, Bmf, Bik and Hrk, serve as sensor proteins of apoptosis, and they induce apoptosis by activating Bak/Bax\u003csup\u003e4\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn healthy hepatocytes, Bcl-xL and Mcl-1 are crucial antiapoptotic proteins, and we previously reported that hepatocyte-specific Mcl-1 knockout (KO) (\u003cem\u003eMcl-1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eAlb-Cre\u003c/em\u003e) mice (Mcl-1\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e mice) and hepatocyte-specific Bcl-xL KO (\u003cem\u003eBcl-xL\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eAlb-Cre\u003c/em\u003e) mice (Bcl-xL\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e mice) exhibit spontaneous hepatocyte apoptosis\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Moreover, knockout of Bak or Bax abolished hepatocyte apoptosis in these mice\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, suggesting that hepatocyte apoptosis induced by deletion of Mcl-1 or Bcl-xL causes autoactivation of Bak/Bax and leads to hepatocyte apoptosis\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. We previously reported that among the eight BH3-only proteins Bid and Bim were functionally active and that disruption of Bid and Bim decreased hepatocyte apoptosis in Bcl-xL\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e mice and Mcl-1\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e mice\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. However, the involvement of all eight BH3-only proteins in maintaining hepatocyte integrity in the healthy liver remains to be elucidated. In the present study, we investigated the comprehensive involvement of all eight BH3-only proteins in Bak/Bax-dependent hepatocyte apoptosis in the absence of antiapoptotic Bcl-2 family proteins.\u003c/p\u003e \u003cp\u003eWe demonstrated that among the eight BH3-only proteins, Puma and Noxa, together with Bid and Bim, are essential regulators that promote Bak/Bax-dependent hepatocyte apoptosis in vivo. Our present study revealed the orchestrated role of BH3-only proteins in maintaining hepatocyte integrity in the healthy liver via Bak/Bax-dependent apoptosis.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eDisruption of Puma suppresses hepatocyte apoptosis in mice with hepatocyte-specific knockout of Bcl-xL or Mcl-1.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe initially examined the role of another BH3-only protein, Puma, in Bak/Bax activation in the absence of Bcl-xL or Mcl-1. We crossed Mcl-1\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e mice and Bcl-xL\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e mice with Puma KO (\u003cem\u003ePuma\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) mice (Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice), and generated Mcl-1\u003csup\u003eΔHep/Δhep\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice and Bcl-xL\u003csup\u003eΔhep/Δhep\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. The expression levels of Mcl-1 and Puma were reduced in the liver tissue of Mcl-1\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Similarly, Bcl-xL and Puma expression in the livers of Bcl-xL\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice was reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Mcl-1\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice and Bcl-xL\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice displayed significantly lower serum alanine transaminase (ALT) levels and reduced serum caspase 3/7 activity than Mcl-1\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e mice and Bcl-xL\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e mice, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). These mice also showed a smaller number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive hepatocytes. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDisruption of Bid, Bim and Puma cannot fully suppress hepatocyte apoptosis induced by the deletion of Bcl-xL and Mcl-1.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe have previously demonstrated that Bcl-xL\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eΔHep/+\u003c/sup\u003e mice, as well as Bcl-xL\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e mice, exhibit impaired liver development, leading to mortality within one day after birth\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Disruption of both Bid and Bim or Bid, Bim and Puma reduced the mortality of Bcl-xL\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eΔHep/+\u003c/sup\u003e mice but had no effect on that of Bcl-xL\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eΔHep/ ΔHep\u003c/sup\u003e mice (\u003cb\u003eTable\u0026nbsp;1, Table\u0026nbsp;2\u003c/b\u003e). Surviving Bcl-xL\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eΔHep/+\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice exhibited hepatocyte apoptosis with elevated serum ALT levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Further disruption of Puma significantly reduced the serum ALT levels, serum caspase 3/7 activity and number of TUNEL-positive hepatocytes in Bcl-xL\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eΔHep/+\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). However, the serum ALT levels in Bcl-xL\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eΔHep/+\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were still significantly greater than those in Bcl-xL\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eΔHep/+\u003c/sup\u003e Bax\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e Bak\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe generated tamoxifen-inducible Bcl-xL\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, in which Bcl-xL and Mcl-1 expression are abolished upon tamoxifen injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), since the knockout of both Bcl-xL and Mcl-1 severely impairs liver development\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e and no Bcl-xL\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice survive after birth (\u003cb\u003eTable\u0026nbsp;2\u003c/b\u003e). After tamoxifen injection for three consecutive days, compared with control mice, Bcl-xL\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice exhibited increased serum ALT levels, elevated caspase 3/7 activity and a higher number of TUNEL-positive hepatocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-H). All mice in the Bcl-xL\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e group were euthanized within 4 days after tamoxifen injection (data not shown). On the other hand, Bcl-xL\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bax\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bak\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice showed no increase in serum ALT levels after tamoxifen injection compared to those without tamoxifen injection (\u003cb\u003eSuppl. Figure\u0026nbsp;1A, 1B)\u003c/b\u003e, and all of these mice survived to day 6 after tamoxifen injection (\u003cb\u003eSuppl. Figure\u0026nbsp;1C)\u003c/b\u003e. These findings suggested that another BH3-only protein, in addition to Bid, Bim and Puma, might activate Bak/Bax and induce hepatocyte apoptosis in the absence of both Bcl-xL and Mcl-1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNoxa knockdown suppresses apoptosis in Bcl-xL-, Mcl-1-, Bid-, Bim- and Puma-deficient hepatocytes.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe generated a doxycycline-inducible Bcl-xL\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e hepatocyte cell line using immortalized primary mouse hepatocytes whose Bcl-xL and Mcl-1 expression was abolished 48 hours after incubation with doxycycline (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Caspase 3/7 activity and LDH activities were significantly increased, and the relative cell viability was significantly decreased after incubation with doxycycline (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Among the five remaining BH3-only proteins, \u003cem\u003eNoxa\u003c/em\u003e, \u003cem\u003eBad\u003c/em\u003e, and \u003cem\u003eBmf\u003c/em\u003e were expressed in this immortalized cell line, while \u003cem\u003eBik\u003c/em\u003e and \u003cem\u003eHrk\u003c/em\u003e were not detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). After doxycycline treatment, only Noxa expression increased among Noxa, Bad and Bmf (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). SiRNA-mediated knockdown of \u003cem\u003eNoxa\u003c/em\u003e led to a significant reduction in Caspase 3/7 activity, LDH activities, and Annexin V-positive areas and a significant increase in the relative cell viability in doxycycline-treated Bcl-xL\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, \u003cb\u003eSuppl. Figure\u0026nbsp;2A\u003c/b\u003e), whereas siRNA-mediated knockdown of \u003cem\u003eBad\u003c/em\u003e or \u003cem\u003eBmf\u003c/em\u003e did not (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, \u003cb\u003eSuppl. Figure\u0026nbsp;2B, 2C\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDisruption of Noxa decreases hepatocyte apoptosis in Bcl-xL Mcl-1-, Bid-, Bim- and Puma-deficient mice and Mcl-1-deficient mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe disrupted Noxa using CRISPR-Cas9 technology to generate tamoxifen-inducible Bcl-xL\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Noxa\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cb\u003eSuppl. Figure\u0026nbsp;3A, 3B\u003c/b\u003e). Disruption of Noxa significantly reduced the serum ALT levels and caspase 3/7 activity in tamoxifen-treated Bcl-xL\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The number of TUNEL-positive hepatocytes was also significantly decreased by Noxa disruption (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Although some Bcl-xL\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Noxa\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice died 5 days after tamoxifen injection, Noxa disruption significantly prolonged their survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo discern the potential contribution of Noxa to hepatocyte apoptosis, particularly in the presence of Bid, Bim and Puma, we knocked out Noxa in Mcl-1\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Compared with Noxa+/+ mice, Noxa\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice showed no differences in serum ALT levels, serum caspase 3/7 activity or number of TUNEL-positive cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Compared with Mcl-1\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e mice, Mcl-1\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e Noxa\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice exhibited significantly lower serum ALT levels and serum caspase 3/7 activity as well as fewer TUNEL-positive hepatocytes in liver sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eNo BH3-only proteins other than Bid, Bim, Puma and Noxa contribute to hepatocyte apoptosis caused by the deletion of both Mcl-1 and Bcl-xL.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo examine the role of the remaining BH3-only proteins in hepatocyte apoptosis caused by the deletion of both Mcl-1 and Bcl-xL, we generated a doxycycline-inducible Bcl-xL\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Noxa\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e hepatocyte cell line using immortalized primary mouse hepatocytes. Caspase 3/7 activity and LDH activities were significantly increased, and the relative cell viability was significantly decreased after incubation with doxycycline (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Among the four remaining BH3-only proteins, \u003cem\u003eBad\u003c/em\u003e and \u003cem\u003eBmf\u003c/em\u003e were expressed in Bcl-xL\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Noxa\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e cells. After doxycycline treatment, \u003cem\u003eBad\u003c/em\u003e and \u003cem\u003eBmf\u003c/em\u003e mRNA expression levels increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). However, siRNA-mediated knockdown of either of these genes did not affect caspase 3/7 activity, LDH activities or relative cell viability in doxycycline-treated Bcl-xL\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Noxa\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). On the other hand, siRNA-mediated knockdown of both \u003cem\u003eBak\u003c/em\u003e and \u003cem\u003eBax\u003c/em\u003e completely abrogated the increases in caspase 3/7 activity and LDH activities and decreased cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo explore other potential activators outside the realm of BH3-only proteins, we performed RNA-seq of a doxycycline-induced Bcl-xL\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Noxa\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e immortalized hepatocyte cell line and a doxycycline-induced Bcl-xL\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Noxa\u003csup\u003e+/+\u003c/sup\u003e immortalized hepatocyte cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). RNA-seq analysis revealed 977 genes in Bcl-xL\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Noxa\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e hepatocytes with FPKM values greater than twice those in Bcl-xL\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Noxa\u003csup\u003e+/+\u003c/sup\u003e hepatocytes (\u003cb\u003eSuppl. Table\u0026nbsp;1\u003c/b\u003e). Among these genes, no Bcl-2 family proteins were detected, while \u003cem\u003eCaspase6\u003c/em\u003e, \u003cem\u003eCaspase9\u003c/em\u003e and \u003cem\u003eCaspase12\u003c/em\u003e, which are associated with apoptosis\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, were detected. In the present study, we focused on \u003cem\u003ecaspase6\u003c/em\u003e, which is known as an executor caspase, as well as caspase3/caspase7. SiRNA-mediated knockdown of \u003cem\u003ecaspase6\u003c/em\u003e slightly but significantly increased relative cell viability in doxycycline-treated Bcl-xL\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Noxa\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e cells, while it did not affect caspase-3/7 activity (\u003cb\u003eSuppl. Figure\u0026nbsp;4A, 4B\u003c/b\u003e). Similarly, siRNA-mediated knockdown of \u003cem\u003ecaspase6\u003c/em\u003e slightly increased the relative viability of BNL.CL.2 cells, a murine hepatocyte cell line, treated with ABT-737, a Bcl-xL inhibitor that induces Bak/Bax-dependent hepatocyte apoptosis\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e without affecting caspase-3/7 activity (\u003cb\u003eSuppl. Figure\u0026nbsp;5A\u003c/b\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWhile the Bak/Bax-dependent mitochondrial pathway of apoptosis by Bcl-2 family proteins is precisely analyzed in vitro\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, which BH3-only proteins among Bid, Bim, Puma, Noxa, Bad, Bik, Bmf and Hrk contribute to hepatocyte Bak/Bax activation in the healthy liver remains to be clarified. In the present study, we demonstrated that not only Bim and Bid but also Puma and Noxa exist in a functionally activated state under physiological conditions, collaboratively inducing Bak/Bax-dependent hepatocyte apoptosis when anti-apoptotic Bcl-2 family proteins in hepatocytes are deficient in vivo. Overall, hepatocyte integrity is maintained through the coordination of antiapoptotic bcl-2 family proteins with Bak/Bax activation mediated by Bid, Bim, Puma, and Noxa among the eight BH3-only proteins.\u003c/p\u003e \u003cp\u003eWe demonstrated that disruption of Puma attenuated hepatocyte apoptosis induced by the absence of Bcl-xL or Mcl-1 in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). This result suggested that Puma induces hepatocyte Bak/Bax activation, which is consistent with previous reports in vitro or in mouse embryonic fibroblasts (MEFs)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Additionally, we showed that disruption of Noxa significantly reduced hepatocyte apoptosis induced by the absence of Mcl-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). In Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, additional Noxa knockout mitigated hepatocyte apoptosis induced by the deletion of Bcl-xL and Mcl-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results revealed that Noxa also plays a distinctive role in inducing hepatocyte apoptosis. Notably, there has been no previous in vivo study on the role of Puma and Noxa in hepatocyte Bak/Bax-dependent apoptosis in the healthy liver, and for the first time, we clarified their roles using an in vivo model.\u003c/p\u003e \u003cp\u003eTo explore additional activators beyond Bid, Bim, Puma and Noxa, we analyzed RNA-seq data, and we focused on caspase6 in the present study (\u003cb\u003eSuppl. Table\u0026nbsp;1\u003c/b\u003e). We showed that caspase6 is involved in hepatocyte apoptosis following Bak/Bax activation (\u003cb\u003eSuppl. Figure\u0026nbsp;4A, 4B, Suppl. Figure\u0026nbsp;5A\u003c/b\u003e). It has been reported that caspase6 mediates a positive feedback loop to sustain the caspase cascade in hepatocytes via the AMPK-caspase6 pathway in nonalcoholic steatohepatitis\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, although this feedback loop is not consistent with our findings because caspase6 knockdown had no effect on caspase-3/7 activity (\u003cb\u003eSuppl. Figure\u0026nbsp;4B, 5A\u003c/b\u003e). Another report indicated that caspase-3, -6 and \u0026minus;\u0026thinsp;7 have nonredundant roles and that caspase6 operates downstream of caspase3, contributing partially to the induction of apoptosis\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The results of our study aligns with this finding. Further investigation is required to determine whether caspase6 partially regulates Bak/Bax-dependent hepatocyte apoptosis.\u003c/p\u003e \u003cp\u003eIn the present study, Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Noxa\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice still exhibited hepatocyte apoptosis after the knockout of Bcl-xL and Mcl-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), and these mice exhibited markedly elevated serum ALT levels, increased caspase 3/7 activity and numerous TUNEL-positive hepatocytes on day 5 after tamoxifen injection (data not shown). However, the majority of these mice survived for 5 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). To further confirm the mode of cell death in Bcl-xL- and Mcl-1-deficient hepatocytes in vivo, we generated Bcl-xL\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bax\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bak\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. Tamoxifen injection did not increase the serum ALT levels of these mice compared to those without tamoxifen injection (\u003cb\u003eSuppl. Figure\u0026nbsp;1A)\u003c/b\u003e, indicating that Bax/Bak deficiency completely blocked the hepatocyte apoptosis induced by Mcl-1/Bcl-xL deficiency. Notably, Bcl-xL\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bax\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bak\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice without tamoxifen injection had higher serum ALT levels than Bcl-xL\u003csup\u003e+/+\u003c/sup\u003e Mcl-1\u003csup\u003e+/+\u003c/sup\u003e Bax\u003csup\u003e+/+\u003c/sup\u003e Bak\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. This is most likely due to the leakage of Cre\u003csup\u003eERT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003c/sup\u003e as the expression levels of Bcl-xL and Bak were lower in Bcl-xL\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bax\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bak\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice than in Bcl-xL\u003csup\u003e+/+\u003c/sup\u003e Mcl-1\u003csup\u003e+/+\u003c/sup\u003e Bax\u003csup\u003e+/+\u003c/sup\u003e Bak\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice even without tamoxifen injection (\u003cb\u003eSuppl. Figure\u0026nbsp;1B)\u003c/b\u003e. Moreover, all Bcl-xL\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bax\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bak\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice survived for 6 days after tamoxifen injection, similar to control mice (Bcl-xL\u003csup\u003e+/+\u003c/sup\u003e Mcl-1\u003csup\u003e+/+\u003c/sup\u003e Bax\u003csup\u003e+/+\u003c/sup\u003e Bak\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) (\u003cb\u003eSuppl. Figure\u0026nbsp;1C\u003c/b\u003e), while Bcl-xL\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Mcl-1\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Noxa\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice died by day 6 after tamoxifen injection (data not shown). These findings demonstrate that knocking out Bcl-xL and Mcl-1, even in the absence of the BH3-only proteins Bid, Bim, Puma and Noxa, still induces Bak/Bax-dependent hepatocyte apoptosis in vivo. A previous in vitro study suggested that Bak and Bax are automatically activated when both Bcl-xL and Mcl-1 are blocked in the absence of BH3-only proteins\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. This mechanism could explain why Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Noxa\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice still exhibited hepatocyte apoptosis after the knockout of Bcl-xL and Mcl-1 in the present study.\u003c/p\u003e \u003cp\u003eIn conclusion, we elucidated the orchestrated regulation of Bcl-2 family proteins in hepatocyte apoptosis within a healthy liver, highlighting the roles of the BH3-only proteins Bid, Bim, Puma and Noxa. No other BH3-only proteins other than Bid, Bim, Puma and Noxa are involved in the Bak/Bax-dependent hepatocyte apoptosis induced by Bcl-xL/Mcl-1 deficiency. Understanding the orchestration of these Bcl-2 family proteins in hepatocytes provides insights into hepatocyte homeostasis and pathogenesis related to hepatocyte apoptosis.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eMice\u003c/h2\u003e\n \u003cp\u003ePuma knockout mice (\u003cem\u003ePuma\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e; Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice\u003cem\u003e)\u003c/em\u003e and Bim knockout mice (\u003cem\u003eBim\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e; Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Bid knockout mice (\u003cem\u003eBid\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e; Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice) were generated as described previously\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Bak/Bax double knockout mice (\u003cem\u003eBax\u003c/em\u003e \u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eAlb-Cre Bak\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e; Bax\u003csup\u003e\u0026Delta;Hep/\u0026Delta;Hep\u003c/sup\u003e Bak\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice) were generated as described previously\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Hepatocyte-specific Bcl-xL knockout mice (\u003cem\u003eBcl-xL\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eAlb-Cre\u003c/em\u003e; Bcl-xL\u003csup\u003e\u0026Delta;Hep/\u0026Delta;Hep\u003c/sup\u003e mice)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e and hepatocyte-specific Mcl-1 knockout mice \u003cem\u003e(Mcl-1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eAlb-Cre\u003c/em\u003e; Mcl-1\u003csup\u003e\u0026Delta;Hep/\u0026Delta;Hep\u003c/sup\u003e mice)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e were crossed with Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice to produce Bcl-xL\u003csup\u003e\u0026Delta;Hep/\u0026Delta;Hep\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice and Mcl-1\u003csup\u003e\u0026Delta;Hep/\u0026Delta;Hep\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, respectively. Mated \u003cem\u003eBcl-xL\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eAlb-Cre Puma\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eBcl-xL\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003ePuma\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eMcl-1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eAlb-Cre Puma\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eMcl-1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003ePuma\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u003c/em\u003e\u003c/sup\u003e offspring were analyzed at 6 to 8 weeks of age. We further crossed Bcl-xL\u003csup\u003e\u0026Delta;Hep/\u0026Delta;Hep\u003c/sup\u003e mice, Mcl-1\u003csup\u003e\u0026Delta;Hep/\u0026Delta;Hep\u003c/sup\u003e mice, Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice and Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice to generate Bcl-xL\u003csup\u003e\u0026Delta;Hep/\u0026Delta;Hep\u003c/sup\u003e Mcl-1\u003csup\u003e\u0026Delta;Hep/+\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. The offspring of \u003cem\u003eBcl-xL\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eMcl-1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/+\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eAlb-Cre Bid\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eBim\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e (or \u003cem\u003eBim\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) \u003cem\u003ePuma\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and \u003cem\u003eBcl-xL\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eMcl-1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eBid\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eBim\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e (or \u003cem\u003eBim\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) \u003cem\u003ePuma\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice were analyzed at 6 to 8 weeks of age. Of note, for this mating, we used \u003cem\u003eBim\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e male and \u003cem\u003eBim\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e female mice because \u003cem\u003eBim\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e female mice are infertile. We also crossed Bcl-xL\u003csup\u003e\u0026Delta;Hep/\u0026Delta;Hep\u003c/sup\u003e mice, Mcl-1\u003csup\u003e\u0026Delta;Hep/\u0026Delta;Hep\u003c/sup\u003e mice with Bax\u003csup\u003e\u0026Delta;Hep/\u0026Delta;Hep\u003c/sup\u003e Bak\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice to generate Bcl-xL\u003csup\u003e\u0026Delta;Hep/\u0026Delta;Hep\u003c/sup\u003e Mcl-1\u003csup\u003e\u0026Delta;Hep/+\u003c/sup\u003e Bax\u003csup\u003e\u0026Delta;Hep/\u0026Delta;Hep\u003c/sup\u003e Bak\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. The offspring of \u003cem\u003eBcl-xL\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eMcl-1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/+\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eBax\u003c/em\u003e \u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eAlb-Cre Bak\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and \u003cem\u003eBcl-xL\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eMcl-1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eBax\u003c/em\u003e \u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eBak\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice were analyzed at 6 to 8 weeks of age. Tamoxifen-inducible hepatocyte-specific Cre mice (\u003cem\u003eAlbumin-Cre-ERT2\u003c/em\u003e) were kindly provided by Professor Pierre Chambon\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, and we generated tamoxifen-inducible hepatocyte-specific Bcl-xL and Mcl-1 knockout mice (\u003cem\u003eBcl-xL\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eMcl-1\u003c/em\u003e \u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eAlb- Cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e; Bcl-xL\u003csup\u003ei\u0026Delta;Hep/i\u0026Delta;Hep\u003c/sup\u003e Mcl-1\u003csup\u003ei\u0026Delta;Hep/i\u0026Delta;Hep\u003c/sup\u003e mice) and Bcl-xL\u003csup\u003ei\u0026Delta;Hep/i\u0026Delta;Hep\u003c/sup\u003e Mcl-1\u003csup\u003ei\u0026Delta;Hep/i\u0026Delta;Hep\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice by crossing them. We also generated Bcl-xL\u003csup\u003ei\u0026Delta;Hep/i\u0026Delta;Hep\u003c/sup\u003e Mcl-1\u003csup\u003ei\u0026Delta;Hep/i\u0026Delta;Hep\u003c/sup\u003e Bax\u003csup\u003ei\u0026Delta;Hep/i\u0026Delta;Hep\u003c/sup\u003e Bak\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice by crossing them with Bax\u003csup\u003e\u0026Delta;Hep/\u0026Delta;Hep\u003c/sup\u003e Bak\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. The mice were injected intraperitoneally with 1 mg of tamoxifen (Sigma‒Aldrich, St. Louis, MO, USA) at the indicated times. The animals were housed in cages under specific pathogen-free conditions with free access to water and standard mouse chow.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eGeneration of Noxa knockout mice\u003c/h2\u003e\n \u003cp\u003eFertilized eggs from \u003cem\u003eBcl-xL\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eMcl-1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eAlb-Cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eBid\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eBim\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003ePuma\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003emice\u003c/em\u003e were produced and used for in vitro fertilization. Subsequently, Noxa knockout guide RNA (gRNA) and Cas9 proteins were introduced into these fertilized eggs through electroporation. gRNA was designed using a software tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crispor.tefor.net/and\u003c/span\u003e\u003c/span\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://crispr.dbcls.jp/\u003c/span\u003e\u003c/span\u003e) to predict unique target sites throughout the mouse genome. Cas9 proteins were obtained from Alt-R\u0026reg; S.p.Cas9 Nucleases 3NLS (Integrated DNA Technologies, Inc., USA). Deletion of the Noxa sequence was confirmed by genotyping DNA isolated from the resulting offspring (\u003cstrong\u003eSuppl. Figure\u0026nbsp;3A, 3B\u003c/strong\u003e). We generated Bcl-xL\u003csup\u003ei\u0026Delta;Hep/i\u0026Delta;Hep\u003c/sup\u003e Mcl-1\u003csup\u003ei\u0026Delta;Hep/i\u0026Delta;Hep\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Noxa\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice by crossing \u003cem\u003eBcl-xL\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eMcl-1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eAlb-Cre\u003c/em\u003e\u003csup\u003e\u003cem\u003eERT\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eBid\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eBim\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003e(or Bim\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e) Puma\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eNoxa\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice with \u003cem\u003eBcl-xL\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eMcl-1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eBid\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eBim\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003e(or Bim\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e) Puma\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eNoxa\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice and analyzed their offspring.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eQuantitative real-time RT‒PCR analysis\u003c/h2\u003e\n \u003cp\u003eTotal RNA was extracted from cells and liver tissues by using an RNeasy Kit (QIAGEN, Hilden, Germany) and reverse transcribed using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan). Real-time RT‒PCR was performed by using TaqMan gene expression assays with an HT7900 Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The following TaqMan gene expression primers were used: \u003cem\u003eNoxa\u003c/em\u003e (Mm00451763_m1), \u003cem\u003eBad\u003c/em\u003e (Mm00432042_m1), \u003cem\u003eBmf\u003c/em\u003e (Mm00506773_m1), \u003cem\u003eBik\u003c/em\u003e (Mm00476123_m1), \u003cem\u003eHrk\u003c/em\u003e (Mm01208086_m1), \u003cem\u003eBax\u003c/em\u003e (Mm00432051_m1), \u003cem\u003eBak\u003c/em\u003e (Mm00432045_m1), \u003cem\u003eCaspase6\u003c/em\u003e (Mm01321726_g1) and \u003cem\u003eActb\u003c/em\u003e (Mm02619580_g1). Gene expression levels were normalized to those of \u003cem\u003eActb\u003c/em\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eWestern blotting analysis\u003c/h2\u003e\n \u003cp\u003eWhole-cell extracts and liver tissues were lysed in lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, phosphate-buffered saline, pH 7.4, 0.1% sodium dodecyl sulfate, 1\u0026times; proteinase inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and 1\u0026times; phosphatase inhibitor cocktail (Nacalai Tesque). The supernatant was collected by centrifugation (13,500 \u0026times; \u003cem\u003eg\u003c/em\u003e for 30 min at 4\u0026deg;C), and the protein concentrations were determined by a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Protein samples of equal concentration were separated on sodium dodecyl sulfate polyacrylamide gels and transferred onto polyvinylidene fluoride membranes. For immunodetection, the following antibodies were used: an anti-Bid antibody (#2003), anti-Bim antibody (#2933), anti-Puma antibody (#24633S), and anti-Bad antibody (#9292) purchased from Cell Signaling Technology (Beverly, MA, USA); an anti-Mcl-1 antibody (600-401-394) purchased from Rockland Immunochemicals (Limerick, PA, USA); an anti-Bcl-xL antibody (sc-634) purchased from Santa Cruz Biotechnology (Dallas, TX, USA); an anti-Noxa antibody (ab23563) and anti-Bmf antibody (ab9655) purchased from Abcam (Cambridge, MA, USA); and an anti-\u0026beta;-actin antibody (A5316) purchased from Sigma‒Aldrich.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eSmall interfering RNA (siRNA)-mediated knockdown\u003c/h2\u003e\n \u003cp\u003eImmortalized cells were transfected with 10 nM siRNA by Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s protocol. The cells were treated with 0.3 \u0026micro;M doxycycline (Wako Pure Chemical Industries, Osaka, Japan) for 24 hours and then analyzed 72 hours after transfection. The following siRNAs were used: siRNA against \u003cem\u003eNoxa\u003c/em\u003e (s81669), siRNA against \u003cem\u003eBad\u003c/em\u003e (s233500), siRNA against \u003cem\u003eBmf\u003c/em\u003e (s101192), siRNA against \u003cem\u003eBak\u003c/em\u003e (s62860), siRNA against \u003cem\u003eBax\u003c/em\u003e (s62874), and siRNA against \u003cem\u003eCaspase6\u003c/em\u003e (s63387). The appropriate negative controls were purchased from Thermo Fisher Scientific.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eHepatic function, caspase activity, and hepatocyte death analyses\u003c/h2\u003e\n \u003cp\u003eSerum alanine transaminase (ALT) levels were measured by using a DRI-CHEM NX700iV (FUJIFILM, Tokyo, Japan). Serum caspase 3/7 activity was measured with a luminescent substrate assay for caspase-3 and caspase-7 (Caspase-Glo Assay, Promega, Madison, WI, USA) according to the manufacturer\u0026rsquo;s protocol and is shown as relative values. Liver sections were stained with hematoxylin and eosin (HE). To detect apoptotic hepatocytes, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was performed with an ApopTag Kit (Millipore, Moldheim, France) according to the manufacturer\u0026rsquo;s protocol. TUNEL-positive cells were counted in four fields per liver section, and the average number of TUNEL-positive cells was determined.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eIn vitro cell death assay\u003c/h2\u003e\n \u003cp\u003eCell viability was measured by a water-soluble tetrazolium salt (WST) assay (Nakalai Tesque), and the results are shown as relative values. Caspase3/7 activity was measured with a luminescent substrate assay for caspase-3 and caspase-7 (Promega) according to the manufacturer\u0026rsquo;s protocol and is shown as relative values. The LDH activities of cultured cells was measured using a Cytotoxicity LDH Assay Kit-WST (Dojindo Laboratories, Kumamoto, Japan), and the results are shown as relative values. ABT-737 was purchased from Selleck Chemicals (Houston, TX, USA). An IncuCyte SX1 live-cell analysis system (Sartorius Japan, Tokyo, Japan) was used to analyze the apoptosis of immortalized cells cultured with doxycycline. Twenty-four hours after siRNA-mediated knockdown, Annexin V Green Reagent for Apoptosis (Sartorius, Japan) was added to the culture medium of the immortalized cells at the same time as doxycycline. Images of Annexin V-positive areas at different time points were obtained and the fluorescence intensity was normalized to that of the Annexin V-positive areas at 0 hours. All of the images were analyzed by using IncuCyte 2021A software (Sartorius Japan).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003ePrimary hepatocyte culture, isolation and immortalization\u003c/h2\u003e\n \u003cp\u003eMouse primary hepatocytes were isolated using the two-step collagenase-pronase liver perfusion method in the same manner as previously reported\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Isolated hepatocytes were cultured in William\u0026rsquo;s Eagle medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FCS), 2 mM L-glutamine (Thermo Fisher Scientific), 100 nM insulin (Sigma‒Aldrich) and 100 nM dexamethasone (Sigma‒Aldrich). To immortalize mouse primary hepatocytes, they were transfected with a lentiviral vector expressing simian virus 40 large T antigen (SV40T) purchased from Addgene (Watertown, MA, USA, plasmid #22298). BNL CL.2 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).\u003c/p\u003e\n \u003cp\u003eThese cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM; Sigma‒Aldrich) in an incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. The medium contained 10% fetal bovine serum and 1% antibiotics (Anti-Anti; Thermo Fisher).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eGeneration of immortalized cells with doxycycline-dependent Cre/LoxP recombination\u003c/h2\u003e\n \u003cp\u003eThe plasmid (pLenti-Cre-IRES-PuroR, Addgene #30205) was amplified by PCR and electrophoresed on a 0.8% gel. The 1029 bp band was eluted from the gel and used as an insert. The vector (pLenti-iCas9-neo, Addgene #85400) was cleaved by two restriction enzymes, XhoI and BsmBI (Bio-Rad, Hercules, CA, USA), and electrophoresed on a 0.8% gel. The 9959 bp band was eluted and used as a backbone. Using an In-Fusion cloning kit (Takara Bio Inc., Shiga, Japan) with these inserts and backbones, a plasmid with doxycycline-inducible Cre and neomycin resistance gene constructs (pLenti-iCre-neo) was prepared. The obtained plasmids were transfected into HEK293 cells for lentivirus packaging. The viral supernatant was used to transfect the immortalized primary hepatocytes. These cells were cultured with 700 \u0026micro;g/ml neomycin (Thermo Fisher Scientific) for 1 week, and pLenti-iCre-Neo-transfected immortalized cells were selected. Transfected immortalized cells were then subjected to the limiting dilution method to generate a monoclonal stable cell line.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eStatistical analysis was performed using GraphPad Prism 9.4.0 (GraphPad, La Jolla, CA, USA). Two-tailed unpaired \u003cem\u003et\u003c/em\u003e tests were used to compare the differences between two groups. One-way ANOVA with Sidak\u0026rsquo;s multiple comparisons test or Dunnett\u0026apos;s multiple comparisons test was performed to compare multiple groups. The Kaplan‒Meier method was used for survival analysis. A \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate statistical significance. All of the data are expressed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SDs. All of the in vitro experiments were repeated at least three times unless otherwise indicated.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupported by Grants-in-Aid for Scientific Research from the Japan Agency for Medical Research and Development (JP23fk0210121 and JP23fk0310512 to T. Takehara) and Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, Japan (JP21H02903 to T. Takehara and JP23H02894 to H.H.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data will be made available immediately after publication on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our gratitude to Dr. Yuko Kotani and their colleagues from The Institute of Experimental Animal Sciences, Faculty of Medicine, Osaka University, for generating Noxa knockout mice using CRISPR-Cas9 technology and Mayumi Honma at Osaka University Graduate School of Medicine for performing the outstanding immunohistochemistry experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepartment of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan\u003c/p\u003e\n\u003cp\u003eShinnosuke Kudo\u003csup\u003e§\u003c/sup\u003e, Hayato Hikita, Yoshinobu Saito\u003csup\u003e§\u003c/sup\u003e, Kazuhiro Murai, Takahiro Kodama, Tomohide Tatsumi, Tetsuo Takehara\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e§\u003c/sup\u003eThese authors contributed equally: Shinnosuke Kudo, Yoshinobu Saito\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.K. and Y.S. conceived and designed the experiments; generated, analyzed, and interpreted the data; and drafted the manuscript. K.M., T.K. and T. Tatsumi provided conceptual input. H.H. and T. Takehara conceived and oversaw the study, designed the experiments, and drafted and edited the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll of the mice were treated humanely, and all of the animal experiments were approved by the Animal Care and Use Committee of Osaka University Medical School (Osaka, Japan) and the Animal Care Committee of Central Institute for Experimental Animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupported by\u0026nbsp;Grants-in-Aid for Scientific Research from the Japan Agency for Medical Research and Development (JP23fk0210121 and JP23fk0310512 to T. Takehara) and\u0026nbsp;Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, Japan (JP21H02903 to T. Takehara and JP23H02894 to H.H.).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCzabotar PE, Garcia-Saez AJ. Mechanisms of BCL-2 family proteins in mitochondrial apoptosis. Nature Reviews Molecular Cell Biology 2023, 24(10): 732\u0026ndash;748.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh R, Letai A, Sarosiek K. Regulation of apoptosis in health and disease: the balancing act of BCL-2 family proteins. Nat Rev Mol Cell Biol 2019, 20(3): 175\u0026ndash;193.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaloni D, Diepstraten ST, Strasser A, Kelly GL. BCL-2 protein family: attractive targets for cancer therapy. Apoptosis 2023, 28(1\u0026ndash;2): 20\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGlab JA, Mbogo GW, Puthalakath H. BH3-Only Proteins in Health and Disease. International Review of Cell and Molecular Biology, \u003cem\u003eVol 328\u003c/em\u003e 2017, 328: 163\u0026ndash;196.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHikita H, Takehara T, Kodama T, Shimizu S, Hosui A, Miyagi T, \u003cem\u003eet al.\u003c/em\u003e BH3-Only Protein Bid Participates in the Bcl-2 Network in Healthy Liver Cells. Hepatology 2009, 50(6): 1972\u0026ndash;1980.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakehara T, Tatsumi T, Suzuki T, Rucker EB, 3rd, Hennighausen L, Jinushi M, \u003cem\u003eet al.\u003c/em\u003e Hepatocyte-specific disruption of Bcl-xL leads to continuous hepatocyte apoptosis and liver fibrotic responses. Gastroenterology 2004, 127(4): 1189\u0026ndash;1197.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHikita H, Kodama T, Shimizu S, Li W, Shigekawa M, Tanaka S, \u003cem\u003eet al.\u003c/em\u003e Bak deficiency inhibits liver carcinogenesis: A causal link between apoptosis and carcinogenesis. Journal of Hepatology 2012, 57(1): 92\u0026ndash;100.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen HC, Kanai M, Inoue-Yamauchi A, Tu HC, Huang YF, Ren DC, \u003cem\u003eet al.\u003c/em\u003e An interconnected hierarchical model of cell death regulation by the BCL-2 family. Nature Cell Biology 2015, 17(10): 1270-+.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKodama T, Hikita H, Kawaguchi T, Saito Y, Tanaka S, Shigekawa M, \u003cem\u003eet al.\u003c/em\u003e The Bcl-2 homology domain 3 (BH3)-only proteins Bim and bid are functionally active and restrained by anti-apoptotic Bcl-2 family proteins in healthy liver. J Biol Chem 2013, 288(42): 30009\u0026ndash;30018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHikita H, Takehara T, Shimizu S, Kodama T, Li W, Miyagi T, \u003cem\u003eet al.\u003c/em\u003e Mcl-1 and Bcl-xL Cooperatively Maintain Integrity of Hepatocytes in Developing and Adult Murine Liver. Hepatology 2009, 50(4): 1217\u0026ndash;1226.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKesavardhana S, Malireddi RKS, Kanneganti TD. Caspases in Cell Death, Inflammation, and Pyroptosis. Annu Rev Immunol 2020, 38: 567\u0026ndash;595.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakagawa T, Zhu H, Morishima N, Li E, Xu J, Yankner BA, \u003cem\u003eet al.\u003c/em\u003e Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature 2000, 403(6765): 98\u0026ndash;103.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaito Y, Hikita H, Nozaki Y, Kai Y, Makino Y, Nakabori T, \u003cem\u003eet al.\u003c/em\u003e DNase II activated by the mitochondrial apoptotic pathway regulates RIP1-dependent non-apoptotic hepatocyte death via the TLR9/IFN-beta signaling pathway. Cell death and differentiation 2019, 26(3): 470\u0026ndash;486.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKing L, Rodriguez-Enriquez R, Pedley R, Mellor C, Wang P, Zindy E, \u003cem\u003eet al.\u003c/em\u003e Apoptotic priming is defined by the dynamic exchange of Bcl-2 proteins between mitochondria and cytosol. Cell death and differentiation 2022, 29: 2262\u0026ndash;2274.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamazaki T, Galluzzi L. BAX and BAK dynamics control mitochondrial DNA release during apoptosis. Cell death and differentiation 2022, 29: 1296\u0026ndash;1298.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim H, Tu HC, Ren DR, Takeuchi O, Jeffers JR, Zambetti GP, \u003cem\u003eet al.\u003c/em\u003e Stepwise Activation of BAX and BAK by tBID, BIM, and PUMA Initiates Mitochondrial Apoptosis. Molecular Cell 2009, 36(3): 487\u0026ndash;499.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao P, Sun X, Chaggan C, Liao Z, In Wong K, He F, \u003cem\u003eet al.\u003c/em\u003e An AMPK-caspase-6 axis controls liver damage in nonalcoholic steatohepatitis. Science 2020, 367(6478): 652\u0026ndash;660.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSlee EA, Adrain C, Martin SJ. Executioner caspase-3, -6, and \u0026ndash;\u0026thinsp;7 perform distinct, non-redundant roles during the demolition phase of apoptosis. J Biol Chem 2001, 276(10): 7320\u0026ndash;7326.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang K, O'Neill KL, Li J, Zhou W, Han N, Pang XM, \u003cem\u003eet al.\u003c/em\u003e BH3-only proteins target BCL-xL/MCL-1, not BAX/BAK, to initiate apoptosis. Cell Research 2019, 29(11): 942\u0026ndash;952.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYin XM, Wang K, Gross A, Zhao YG, Zinkel S, Klocke B, \u003cem\u003eet al.\u003c/em\u003e Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis. Nature 1999, 400(6747): 886\u0026ndash;891.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchuler M, Dierich A, Chambon P, Metzger D. Efficient temporally controlled targeted somatic mutagenesis in hepatocytes of the mouse. Genesis 2004, 39(3): 167\u0026ndash;172.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 2 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-differentiation","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cdd","sideBox":"Learn more about [Cell Death \u0026 Differentiation](http://www.nature.com/cdd/)","snPcode":"41418","submissionUrl":"https://mts-cdd.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Differentiation","twitterHandle":"@cddpress","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4704982/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4704982/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe fine-tuned balance between anti-apoptotic Bcl-2 family proteins, such as Bcl-xL and Mcl-1, and pro-apoptotic Bcl-2 family proteins, like Bak and Bax, is crucial for maintaining hepatocyte integrity. BH3-only proteins, including Bid, Bim, Puma, Noxa, Bad, Bik, Bmf and Hrk, serve as apoptosis sensors activating Bak and Bax. We previously reported that BH3-only proteins Bid and Bim contribute to hepatocyte apoptosis through Bak/Bax activation in the absence of antiapoptotic proteins, Bcl-xL and/or Mcl-1. However, the comprehensive involvement of all eight BH3-only proteins maintaining hepatocyte integrity in healthy livers remains unclear.\u003c/p\u003e \u003cp\u003ePuma disruption suppressed hepatocyte apoptosis in hepatocyte-specific Bcl-xL or Mcl-1 knockout (Bcl-xL\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e or Mcl-1\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e) mice. Disruption of Bid and Bim partially prevented lethality in Mcl-1\u003csup\u003eΔHep/+\u003c/sup\u003e Bcl-xL\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e mice, although severe hepatocyte apoptosis persisted, which was suppressed by additional Puma disruption. However, hepatocyte apoptosis was still strongly induced compared to that in Mcl-1\u003csup\u003eΔHep/+\u003c/sup\u003e Bcl-xL\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e Bax\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e Bak\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. Triple disruption of Bid, Bim and Puma did not prevent induction of hepatocyte apoptosis in tamoxifen-induced Mcl-1\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bcl-xL\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e mice.\u003c/p\u003e \u003cp\u003ePrimary hepatocytes, isolated from Mcl-1\u003csup\u003efl/fl\u003c/sup\u003e Bcl-xL\u003csup\u003efl/fl\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice and immortalized, underwent apoptosis with doxycycline-dependent Cre recombination. Among the remaining five BH3-only proteins, Bik and Hrk were not expressed in this cell line, and Noxa knockdown, but not Bad or Bmf knockdown, reduced apoptosis. Noxa disruption alleviated hepatocyte apoptosis in Mcl-1\u003csup\u003eΔHep/ΔHep\u003c/sup\u003e mice and tamoxifen-induced Mcl-1\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bcl-xL\u003csup\u003eiΔHep/iΔHep\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, prolonging survival. Apoptosis persisted in immortalized primary hepatocytes isolated from Mcl-1\u003csup\u003efl/fl\u003c/sup\u003e Bcl-xL\u003csup\u003efl/fl\u003c/sup\u003e Bid\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Bim\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Puma\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e Noxa\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice where doxycycline-dependent Cre recombination was induced, but was completely suppressed by Bak/Bax knockdown, while Bad or Bmf knockdown had no effect.\u003c/p\u003e \u003cp\u003eIn conclusion, among the eight BH3-only proteins, Puma and Noxa, alongside Bid and Bim, contributed to sustained Bak/Bax-dependent hepatocyte apoptosis in the absence of Mcl-1 and Bcl-xL, elucidating the orchestration of Bcl-2 family proteins in healthy livers.\u003c/p\u003e","manuscriptTitle":"Collaborative orchestration of BH3-only proteins governs Bak/Bax-dependent hepatocyte apoptosis under antiapoptotic protein-deficiency in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-31 14:05:16","doi":"10.21203/rs.3.rs-4704982/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-08-01T11:07:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-08-01T00:08:53+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-07-18T21:49:10+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-07-17T23:45:01+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-07-17T01:27:40+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-07-16T01:26:28+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-07-15T13:15:20+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-07-08T12:38:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-08T12:32:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-08T10:49:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Differentiation","date":"2024-07-08T10:49:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-differentiation","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cdd","sideBox":"Learn more about [Cell Death \u0026 Differentiation](http://www.nature.com/cdd/)","snPcode":"41418","submissionUrl":"https://mts-cdd.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Differentiation","twitterHandle":"@cddpress","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f1717fe0-2c72-48c0-b8c8-99dc0e8eec68","owner":[],"postedDate":"July 31st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":34283366,"name":"Biological sciences/Cell biology"},{"id":34283367,"name":"Biological sciences/Biochemistry/Proteins"},{"id":34283368,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2025-02-25T08:09:56+00:00","versionOfRecord":{"articleIdentity":"rs-4704982","link":"https://doi.org/10.1038/s41418-025-01458-y","journal":{"identity":"cell-death-and-differentiation","isVorOnly":false,"title":"Cell Death \u0026 Differentiation"},"publishedOn":"2025-02-24 05:00:00","publishedOnDateReadable":"February 24th, 2025"},"versionCreatedAt":"2024-07-31 14:05:16","video":"","vorDoi":"10.1038/s41418-025-01458-y","vorDoiUrl":"https://doi.org/10.1038/s41418-025-01458-y","workflowStages":[]},"version":"v1","identity":"rs-4704982","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4704982","identity":"rs-4704982","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}