Discovery of Hsp70-Mediated Lysosomal Repair as a Highly Specific Target for Senolysis

Discovery of Hsp70-Mediated Lysosomal Repair as a Highly Specific Target for Senolysis | 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 Discovery of Hsp70-Mediated Lysosomal Repair as a Highly Specific Target for Senolysis Sheng Ding, Yan-Lei Fan, Fangfang Jiao, Yang Zhao, Yuxia Zhang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6662488/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Senescent cells (SnCs) play a central role in aging and various age-related diseases, making their selective elimination a promising therapeutic strategy for extending lifespan and mitigating these conditions. However, the development of selective senolytic agents that exclusively target SnCs remains a challenge due to the absence of mechanisms that distinctly differentiate them from non-senescent cells (non-SnCs). In this study, we report a serendipitous discovery that Pifithrin-µ (PES), through its inhibition of Hsp70, exhibits highly selective senolytic activity. Mechanistic investigations revealed that SnCs harbor dysfunctional lysosomes with compromised membrane integrity—a vulnerability typically counteracted by the upregulation and lysosomal recruitment of Hsp70, which facilitates lysosomal repair by enhancing ceramide production. Inhibition of Hsp70 by PES selectively disrupts this repair mechanism, inducing catastrophic lysosomal membrane permeability (LMP) and triggering lysosome-dependent cell death (LDCD) specifically in SnCs. Moreover, PES treatment improved aging-related phenotypes and reversed lung fibrosis in mouse models of irradiation-induced accelerated aging and pulmonary fibrosis, respectively. These findings establish Hsp70 as a novel and transformative senolytic target and suggest that targeting the senescence-specific lysosomal repair pathway could provide a safer, more selective, and efficacious alternative to current senolytic strategies, which primarily target non-specific anti-apoptotic pathways. Biological sciences/Cell biology/Senescence Biological sciences/Cell biology/Mechanisms of disease Biological sciences/Cell biology/Cell death Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction SnCs are significant contributors to aging and a wide range of age-related diseases, including tissue fibrosis, osteoarthritis, Alzheimer’s disease, and cancer 1 – 4 . Cellular senescence can be induced by DNA damage, oncogene activation, or excessive cellular replication 5 . Once senescent, these cells evade apoptosis and secrete pro-inflammatory factors—collectively referred to as the senescence-associated secretory phenotype (SASP) factors—which contribute to tissue dysfunction and accelerate aging and age-related disease progression 6 – 10 . Consequently, targeted elimination of SnCs (senolysis) has emerged as a promising therapeutic strategy for tissue rejuvenation and the treatment of age-related disorders 11 – 19 . Several senolytic compounds, including Navitoclax (ABT-263), Fisetin, and the Dasatinib-Quercetin (D&Q) combination, have shown efficacy in clearing SnCs both in vitro and in vivo 20 . However, their clinical application is limited by poor selectivity and significant adverse effects, largely because they target anti-apoptotic pathways that are also critical for the survival of normal cells 20 , 21 . Additionally, the inherent heterogeneity of SnCs, which is characterized by diverse phenotypes and molecular profiles, further complicates the development of broadly effective senolytic strategies 2 , 5 . Therefore, identifying vulnerabilities that are specific to SnCs and absent in non-SnCs may provide a path toward more selective and effective senolytic approaches. In SnCs, lysosomes have been shown to exhibit reduced acidity and compromised membrane integrity 13 , 22 , 23 . However, identifying a senescent cell-specific mechanism of lysosomal modulation and directly targeting such lysosomal vulnerability in a senescent cell-specific manner remains underexplored. In this study, we serendipitously discovered that PES 24 – 27 , through inhibition of Hsp70, exhibits unprecedented selective senolytic activity by inducing significant cell death exclusively in SnCs while sparing non-SnCs. Mechanistic investigations revealed a previously unrecognized role for Hsp70 in repairing defective lysosomal membranes to maintain SnC survival. This finding is consistent with Hsp70’s role in stabilizing lysosomes in Niemann–Pick disease, where mutations in the acid sphingomyelinase (ASM) gene result in markedly reduced lysosomal stability 28 . Inhibition of Hsp70 by PES disrupts this repair mechanism, inducing extensive LMP and cathepsins release-triggered LDCD, ultimately leading to the selective elimination of SnCs. Moreover, in vivo treatment with PES selectively cleared SnCs, and consequently ameliorated aging phenotypes and reversed lung fibrosis in mouse models of irradiation-induced accelerated aging and pulmonary fibrosis, respectively. These findings establish Hsp70 inhibition as a novel, highly selective, and broadly effective senolytic strategy that targets the lysosomal membrane repair mechanism specifically activated in SnCs, thereby offering a promising therapeutic approach for treating aging and age-related diseases. PES induces selective senolysis via Hsp70 inhibition In our investigation of SnCs, we unexpectedly discovered that treatment with a small molecule, PES, induced significant cell death in senescent A549 cells (an adenocarcinomic human alveolar basal epithelial cell line) while largely sparing non-SnCs (Fig. 1 a,b). To assess whether PES-mediated senolysis is broadly effective across different cell types with senescence induced by distinct mechanisms, we tested its senolytic activity across multiple cell lines, including H1299 (a human non-small cell lung carcinoma cell line, p53-null), MCF10A (a non-tumorigenic human mammary epithelial cell line), and HFF1 (a normal human foreskin fibroblast cell line). Senescence was induced under various conditions: Bleomycin-induced DNA damage (D-SnCs) 18 , Nutlin-3a-induced p53 activation (N-SnCs) 13 , and H 2 O 2 -induced oxidative stress (Ox-SnCs) 29 (Fig. S1 a,b). Notably, treatment with 10 µM PES induced cell death in over 50% of SnCs in all tested cell lines while exerting negligible effects on their corresponding non-SnCs (Fig. 1 c, Fig.S2a-c). Similarly, PES induced selective senolysis in replication-induced senescent mouse embryonic fibroblasts (R-SnCs) compared to their non-senescent counterparts (Fig.S2d). To compare the selectivity of PES-mediated senolysis with that of previously identified senolytic agents, we calculated their selectivity by determining the viability ratio (R), which is the percentage of viable non-SnCs relative to SnCs at the same treatment concentration. Our analysis revealed that PES exhibits selectivity several orders of magnitude greater than previously identified senolytic agents, such as ABT-263, D&Q, and BPTES (Fig. 1 d, Fig.S3a-c). Collectively, these results demonstrate that PES induces senolysis with significantly greater selectivity than other senolytic agents, suggesting that its targeting mechanism is crucial for SnC survival but not for non-SnCs. It has been previously established that PES inhibits Hsp70. To confirm that PES induces selective senolysis through Hsp70 inhibition, we compared its effect with another Hsp70 inhibitor, MKT-077 30 , on SnCs and non-SnCs. Consistent with the PES findings, SnCs exhibited significant cell death upon MKT-077 treatment (Fig.S4a), while non-SnCs remained largely viable. To further confirm the specificity of Hsp70 targeting, shRNA-mediated knockdown of HspA1A (a specific gene that encodes Hsp70 protein) induced selective senolysis, resulting in approximately 50% cell death in SnCs while leaving non-SnCs nearly unaffected (Fig. 1 e). Next, we established a doxycycline-inducible system in SnCs to overexpress either wild-type HspA1A or its mutant form, HspA1A 2 CA , which prevents PES binding by cysteine-to-alanine mutations at positions 574 and 603 24 (Fig. 1 f). Consistent with the expected mechanism of rescuing target inhibition, overexpression of wild-type HspA1A significantly protected SnCs from PES- or MKT-077-induced cell death (Fig. 1 g, Fig.S4b). Furthermore, SnCs overexpressing the PES-resistant mutant HspA1A 2 CA exhibited greater resistance to PES-induced cell death, with over 80% survival at 50 µM PES—a concentration that induced nearly complete cell death in cells overexpressing wild-type HspA1A (Fig. 1 h). Moreover, a small-molecule activator of Heat Shock Transcription Factor 1 (HSF1), HSF1A, which enhances endogenous HspA1A transcription, similarly rescued PES- or MKT-077-induced cell death in SnCs (Fig. 1 i, Fig.S4c). Collectively, these findings confirmed that PES induces selective senolysis through Hsp70 inhibition, leading to significant cell death in SnCs while sparing non-SnCs. Hsp70 Facilitates Lysosomal Membrane Repair in SnCs To elucidate the mechanism by which Hsp70 specifically maintains the survival of SnCs and how this is disrupted by PES, we first examined whether Hsp70 is differentially expressed between SnCs and non-SnCs. qPCR and Western blot analyses revealed that both mRNA and protein levels of Hsp70 were significantly higher in SnCs than in non-SnCs, which was consistent with more pronounced nuclear accumulation of HSF1 in SnCs (Fig.S5a-d). To further characterize Hsp70 subcellular localization, immunostaining revealed that Hsp70 was prominently co-localized with the lysosomal-associated membrane protein 1 (Lamp1), a marker of lysosomes, in SnCs, a feature not observed in non-SnCs (Fig. 2 a). This exclusive lysosomal localization suggests that Hsp70 may play a critical role in lysosomal regulation, a mechanism essential for SnC survival. To explore this hypothesis, we characterized lysosomes in SnCs and non-SnCs. Compared to non-SnCs, SnCs exhibited evident nuclear translocation of TFEB—a master transcription factor driving lysosomal biogenesis—along with a significant increase in the protein expression of Lamp1 and Lamp2A and lysosomal proteases cathepsin B (CTSB) and cathepsin D (CTSD) (Fig.S6a-d). These results indicate that lysosomal biogenesis is enhanced in SnCs compared to non-SnCs. Additionally, SnCs exhibited mild lysosomal membrane disruption, as evidenced by emergence of Galectin-8 (Gal8, a marker of early lysosomal rupture) puncta, reduced acridine orange staining (indicative of decreased lysosomal acidity), and leakage of CTSD into the endonuclear compartment (Fig.S6e-h). Collectively, these findings highlight the accumulation of lysosomes with mildly disrupted membrane integrity as a unique feature of SnCs, unequivocally distinguishing them from non-SnCs. Given that Hsp70 has been previously shown to stabilize lysosomal membrane, particularly in the cells from patients with Niemann–Pick disease 28 , its enrichment at lysosomal membranes in SnCs suggests that it may play a key role in repairing lysosomal membrane in these cells. Consistent with this hypothesis, ASM—a key enzyme recruited to the defective lysosomal membrane by Hsp70 in acidic environment and catalyzing hydrolyzation of bis(monoacylglycero)phosphate (BMP) into ceramide, an essential lipid in membrane repair 31 , 32 —was found to be highly expressed in SnCs compared to non-SnCs (Fig.S7a). Furthermore, ASM exhibited a lysosomal enrichment pattern similar to that of Hsp70 in SnCs, as evidenced by its co-localization with the Lamp1 (Fig.S7b). In line with these observations, intracellular ceramide levels were markedly higher in SnCs relative to non-SnCs (Fig. 2 b,c). To assess whether Hsp70 mediates lysosomal membrane repair in an BMP-dependent manner in SnCs, we compared the effects of HspA1A overexpression in SnCs with that of HspA1A W90F —a mutant containing a Trp90Phe substitution that specifically abolishes the interaction between Hsp70 and BMP without disrupting its chaperone activity 28 . Overexpression of HspA1A or upregulation of its endogenous expression via HSF1A treatment further enhanced ceramide production in SnCs (Fig. 2 d,e, Fig.S8a,b), whereas overexpression of H spA1A W90F failed to elevate ceramide levels (Fig. 2 d,e). Moreover, HspA1A overexpression or HSF1A treatment significantly mitigated lysosomal permeability, as indicated by a marked decrease in both Gal8 puncta and endonuclear mislocalization of CTSD, whereas HspA1A W90F overexpression had no such rescuing effect (Fig. 2 f-h, Fig.S8c,d). Consistently, LysoTracker or acridine orange staining revealed that HspA1A overexpression or HSF1A treatment enhanced lysosomal acidification in SnCs—an effect not achieved by overexpressing HspA1A W90F (Fig. 2 i, Fig.S8e,f). Collectively, these findings demonstrate that Hsp70 is specifically upregulated and recruited to lysosomal membranes in SnCs, but not in non-SnCs, where it facilitates membrane repair by promoting ASM-dependent ceramide production (Fig. 2 j). PES Selectively induces Extensive LMP to Trigger LDCD in SnCs Given our new findings that upregulating and recruiting Hsp70 to lysosome in response to senescence induction can facilitate lysosomal membrane repair in SnCs, we next investigated whether Hsp70 is essential for maintaining lysosomal membrane in these cells, thereby preventing lysosomal membrane disruption to a level that could otherwise compromise cell survival. To test this, we assessed LMP in SnCs following PES treatment using Galectin-3 (Gal3) staining. Gal3, a cytosolic β-galactoside-binding lectin, is recruited to damaged lysosomes and coordinates autophagic responses to irreversible lysosomal injury; its lysosomal localization is a widely accepted marker of severe lysosomal membrane damage 33 – 35 . In PES-treated SnCs, we observed a marked cytosolic translocation of Gal3, which was co-localized with Lamp1 (Fig. 3 a), indicating severe lysosomal membrane disruption. Supporting this, PES treatment also led to a significant reduction in acridine orange staining, which reflects a decrease in lysosomal acidity, along with a sharp increase in the cytosolic mislocalization of lysosomal CTSB and CTSD (Fig. 3 b-d). Notably, these PES-induced changes were observed exclusively in SnCs and not in non-SnCs (Fig.S9a,b), demonstrating that PES selectively induces extensive LMP in SnCs. When overexpressing HspA1A in SnCs, the prominent lysosomal localization of Hsp70 remained unchanged (Fig. 3 e). Nuclear disruption, a characteristic downstream consequence of catastrophic LMP, caused by cathepsin release and subsequent nuclear injury 36 , 37 , was evident in PES-treated SnCs but was almost completely prevented in HspA1A -overexpressing SnCs (Fig. 3 e). In contrast to the lysosomal translocation of Gal3 in PES-treated SnCs, HspA1A overexpression effectively blocked this translocation (Fig. 3 f,g), confirming that PES induces extensive LMP through Hsp70 inhibition. Since severe leakage of lysosomal contents, particularly cathepsins, triggers LDCD, we hypothesized that PES-induced senolysis is driven by LDCD. Consistent with this hypothesis, PES-induced cell death in SnCs was largely prevented by pharmacological inhibition of cathepsins, which are known to block LDCD, but not by apoptosis inhibitors such as ZVF and QVO (Fig. 3 h-j). Collectively, these findings demonstrate that Hsp70 plays a crucial role in SnCs to prevent extensive LMP-induced LDCD, a protective mechanism for SnC survival that is effectively targeted by Hsp70 inhibitors like PES to induce highly selective senolysis. PES Mitigates Accelerated Aging in Irradiated Mice To evaluate the in vivo effects of PES-induced selective senolysis on aging, we established an aging mouse model using whole-body irradiation (WBI) at a sublethal dose—a well-established method to accelerate aging in mice, which has been previously used to assess the anti-aging effects of other senolytics 11 , 19 . One month after exposure to 5 Gy WBI, mice were treated with either PES or the D&Q combination for an additional month, followed by a two-month observation period (Fig. 4 a). Four months post-irradiation, mice developed noticeably greyed fur, a hallmark sign of aging. Notably, PES treatment reversed this change, restoring hair color to nearly that of non-irradiated (naïve) mice, while D&Q treatment resulted in minimal recovery (Fig. 4 b). Further analysis of lung tissues revealed that WBI significantly increased the number of senescence-associated β-galactosidase (SA-β-Gal)-positive cells (Fig. 4 c,d), consistent with previous reports showing similar changes 19 . In contrast, both PES and D&Q treatments cleared most of the SA-β-Gal–positive cells within the lungs, reducing their numbers to levels comparable to those seen in naïve mice (Fig. 4 c,d). In line with these findings, the expression of classical senescence markers—such as p16 INK 4 A , p21, and SASP components—was significantly elevated in the lungs following WBI, but all markers returned to near-baseline levels after treatment with PES or D&Q (Fig. 4 e-g, Fig. S10). Additionally, HspA1A expression in lung tissues increased approximately sevenfold following WBI, consistent with our in vitro findings that SnCs upregulate Hsp70 to facilitate lysosomal repair and maintain survival (Fig. 4 h, Fig.S5c,d). In contrast, treatment with PES or D&Q restored HspA1A expression to levels similar to those observed in naïve mice, confirming their robust efficacy in clearing senescent cells (Fig. 4 h). Functional assessments further showed that PES treatment markedly improved grip strength and treadmill endurance, which had regressed following WBI, exhibiting superior effects compared to D&Q (Fig. 4 i,j). Overall, our findings indicate that PES enables effective clearance of SnCs in vivo, particularly in lung tissue, and significantly surpasses D&Q in mitigating aging-related physiological decline in WBI-accelerated aging mice. This superior efficacy, likely arising from PES’s far higher selectivity for SnCs compared to previously identified senolytic agents, underscores the therapeutic potential of pharmacological Hsp70 inhibition for delaying aging and treating age-related diseases. PES Ameliorates Pulmonary Fibrosis in Mice Pulmonary fibrosis often results from the senescence and exhaustion of alveolar type 2 (AT2) stem cells, which regenerate alveolar type 1 (AT1) cells after lung injury 9 , 38 – 40 . Senescent AT2 cells promote fibroblast infiltration and matrix deposition, impairing gas exchange. Although D&Q have shown promise in alleviating these conditions 9 , 16 , 29 , 41 , 42 , their clinical application is limited by inevitable toxicity due to poor selectivity. PES, a highly selective Hsp70 inhibitor that efficiently clears senescent cells with far greater specificity than existing agents, may provide a safer and more effective senolytic approach for treating pulmonary fibrosis. To test this hypothesis, we first compared the effects of PES and D&Q on bleomycin-induced senescent and non-senescent AT2 cells in vitro . As expected, D&Q exhibited significant toxicity toward non-senescent AT2 cells (Fig. S11). By contrast, PES efficiently eliminated senescent AT2 cells while having negligible impact on the survival of non-senescent AT2 cells, even at high concentrations such as 30 µM (Fig. S11). These findings highlight the superior selectivity of PES-induced senolysis, with no observable toxicity to normal AT2 cells, compared to previously identified senolytic agent D&Q. Next, we established a mouse model of pulmonary fibrosis by administering bleomycin to C57BL/6 mice, inducing senescence in AT2 cells, followed by four consecutive weeks of treatment with either PES or D&Q (Fig. 5 a). Both PES and D&Q treatments significantly reduced the expression of senescence markers—including p16 INK 4 A , p21, and SASP components—in lung tissue to levels nearly identical to those observed in normal mice (Fig. 5 b, Fig. S12a-g). Immunostaining further confirmed that both treatments substantially decreased the number of p16 INK 4 A -positive cells in bleomycin-treated lung tissues (Fig. 5 c, d), bringing them to levels comparable to those seen in normal mice. These results consistently indicate that PES effectively eliminates bleomycin-induced SnCs in the lungs. Pulmonary fibrosis progresses when SASP factors secreted from senescent AT2 cells activate fibroblasts and promote their differentiation into myofibroblasts, leading to the increased synthesis and excessive accumulation of collagen and other extracellular matrix (ECM) components (e.g., α-SMA, fibronectin, and MMPs) 42 , 43 . Immunostaining and qPCR analyses revealed that both PES and D&Q treatments significantly reduced the expression and extracellular accumulation of pivotal ECM components, including collagen I, α-SMA, fibronectin, MMP 10, and MMP 12 (Fig. 5 e-h, Fig. S13). Compared to D&Q, PES was able to reduce extracellular α-SMA and fibronectin accumulation to lower levels and uniquely downregulate MMP 10 and MMP 12 expression (Fig. 5 e-h, Fig. S13). Consistently, CT scans demonstrated that treatment with either agent reduced fibrotic (white) regions in the lungs, indicating a substantial decrease in lung inflammation (Fig. S14). Furthermore, pulmonary fibrosis in bleomycin-treated mice was markedly ameliorated following PES or D&Q treatment, as evidenced by improved alveolar structure and reduced areas of lung parenchymal consolidation (Fig. S15a,b). Importantly, whole-body plethysmography (WBP) demonstrated that PES treatment fully restored bleomycin-impaired pulmonary function to near-normal levels, as indicated by significant improvements in minute volume normalized to body weight (an index of ventilatory efficiency diminished in severe fibrosis), inspiratory and expiratory times (prolonged when lung compliance is reduced), peak flow rates (suppressed in fibrotic lungs), relaxation time (prolonged due to impaired elastic recoil in severe fibrosis), and breathing frequency (reduced in advanced pulmonary fibrosis) (Fig. 5 i-p). In contrast, D&Q treatment only partially improved lung function and did not significantly affect inspiratory and expiratory times, relaxation time, or breathing frequency, reflecting persistent impairment in respiratory cycle, ventilatory efficiency, lung compliance, airway patency, and respiratory rhythm 44 (Fig. 5 i-p). Collectively, these findings demonstrate that PES effectively clears SnCs in the lungs and significantly mitigates pulmonary fibrosis, restoring multiple key aspects of pulmonary function to near-normal levels. Notably, PES outperformed D&Q as a senolytic treatment for pulmonary fibrosis, as evidenced by its superior capacity to exhibit extremely low toxicity to non-senescent AT2 cells and comprehensively improve lung function. Our results further highlight the therapeutic potential of pharmacological Hsp70 inhibition for treating not only pulmonary fibrosis but also a broad range of age-related diseases. Discussion SnCs commonly display lysosomes that are increased in number and size, along with elevated SA-β-Gal activity 2 . In these cells, p16 INK 4 a promotes the nuclear translocation of TFEB by inhibiting CDK4, which upregulates genes involved in lysosomal biogenesis and increases lysosomal quantity 22 . However, despite enhanced lysosomal biogenesis, SnCs also exhibit widespread lysosomal leakage, leading to elevated intralysosomal pH and diminishing hydrolase activity 22 . This compromised membrane integrity allows the leakage of lysosomal contents, such as CTSB and CTSD, into the cytoplasm—and even into the nucleus— as observed in senescent microglial cells 23 , 45 , 46 and in our senescent cell models. To mitigate the detrimental effects of severe LMP—including cathepsins release, uncontrolled degradation of cytosolic proteins, and LDCD 34 , 35 , 37 —SnCs likely activate intrinsic lysosomal repair mechanisms. Although several repair pathways, including Hsp70- and ESCRT-mediated lysosomal membrane repair, have been described in various cell types under specific conditions 47 , the mechanisms underlying lysosomal membrane repair in SnCs remain largely unexplored. In our study, we found that Hsp70—a chaperone that facilitates protein folding and prevents the formation of aberrant protein aggregates—was markedly upregulated and recruited to lysosomal membranes in SnCs, but not in non-SnCs. This selective localization confers Hsp70 with a senescence–specific role: patching damaged lysosomes and preventing catastrophic LMP and downstream LDCD. These mechanistic insights explain the highly selective elimination of SnCs by PES, which targets Hsp70 and spares non-SnCs. Moreover, our findings suggest that the senolytic selectivity of pharmacological Hsp70 inhibitors could be further enhanced through chemical modifications that promote lysosomal enrichment. Such modifications likely minimize the impact on lysosome-independent Hsp70 functions in non-SnCs, improving efficacy while reducing side effects. Notably, we also uncovered a previously unrecognized pathway through which Hsp70 specifically supports the survival of SnCs, distinct from its known roles in other cell types including cancer cells 48 . As aging progresses, the accumulation of SnCs contributes to the increased release of SASP components, creating an inflammatory niche that impairs the regenerative capacity of tissue-resident stem cells and drives the development of age-related diseases 7 , 49 – 51 . Pulmonary fibrosis—a progressive and currently incurable lung disease characterized by irreversible scarring and reduced life expectancy—is primarily driven by the senescence of AT2 cells. Recent studies have shown that senolytic agents such as D&Q and ABT-263 can alleviate pulmonary fibrosis in mouse models, but their safety concerns persist due to the effects on apoptotic regulation in non-SnCs 41 , 42 , 52 . In our study, PES demonstrated significantly higher senolytic selectivity compared to previously identified agents, leading to superior improvements in lung function and fibrosis in a pulmonary fibrosis mouse model. Crucially, PES exhibited negligible toxicity toward non-senescent AT2 cells within a defined concentration range, consistent with preclinical findings showing that PES effectively inhibits cancer cell growth without adverse effects on vital organs 53 , 54 . These findings not only reveal an unprecedentedly high level of senolytic selectivity for PES but also suggest a promising therapeutic approach for rejuvenation and the treatment of age-related conditions. Methods Animal models All animal experiments were conducted in compliance with protocols approved by the Institutional Animal Care and Use Committee of Tsinghua University, Beijing, China. Male C57BL/6 mice used in this study were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were bred and maintained in individually ventilated cages (six mice per cage) under specific-pathogen-free conditions in a controlled environment (temperature: 20–26°C; humidity: 40–60%; 12-hour light/12-hour dark cycle). For the bleomycin-induced idiopathic pulmonary fibrosis experiments, 6–8 week-old mice were anesthetized via inhalation of isoflurane and then received an intranasal administration of 5 mg/kg Bleomycin sulfate (Selleck, S1214) or PBS (phosphate-buffered saline) as a control. The mice were monitored daily, and four weeks post-administration, pulmonary fibrosis developed. The animals were then randomized to initiate treatment. Specifically, dasatinib (5 mg/kg) (MedChemExpress, HY10181) plus quercetin (50 mg/kg) (MedChemExpress, cat.no. HY18085) (D&Q) were administrated by oral gavage every four days for four weeks 41 , 42 , and PES (10 mg/kg) (MedChemExpress, HY10940) was administrated by intraperitoneal injection every two days for four weeks 53 , 54 . In parallel, treatment with vehicle served as a control. After the treatment period, the mice were monitored for an additional four weeks to allow sufficient time for lung lesion repair. At the study's endpoint, pulmonary function tests and micro-CT imaging were performed prior to euthanizing the mice. For molecular and pathological evaluation, lung tissues were collected for RNA and protein extraction. Additionally, tissues were paraffin-embedded for immunofluorescence analysis, H&E (haematoxylin and eosin) staining, and Masson's staining, or frozen in OCT solution for SA-β-Gal staining. For the WBI assay, C57BL/6J mice aged 10–12 weeks were exposed to a sublethal dose of 5 Gy using an X-Ray irradiator (RS 2000 Pro, RadSource, USA). One month post-irradiation, the mice were randomly assigned to receive treatments with dasatinib plus quercetin and PES for SnC clearance, following the dosing regimen described in the bleomycin-induced IPF model. Physical functions were evaluated by measuring Grip Strength and Endurance. Grip Strength was assessed using a Grip Strength Meter, with final values averaged over five trials, while endurance was measured using a Treadmill (model 47300, Ugo Basile). Prior to testing, the mice underwent a one-week treadmill training period to acclimate to the exercise protocol. Cell Culture and Maintenance Multiple cell lines were used in this study, including A549 (human lung carcinoma), H1299 (human non-small cell lung carcinoma), MCF10A (human mammary epithelial), and HFF1 (human foreskin fibroblast), as well as primary mouse embryonic fibroblasts (MEFs) and primary alveolar type 2 (AT2) cells isolated from mouse lung tissue. All cell lines were obtained from reputable suppliers such as the American Type Culture Collection (ATCC, USA). A549 and H1299 cells were cultured in DMEM/F-12(1:1) medium (Gibco, Thermo Fisher Scientific, C11330500BT) supplemented with 10% fetal bovine serum (FBS, Gibco, 16000-044) and 1% penicillin-streptomycin (Pen-Strep, Gibco, 15140-122). MCF10A cells were maintained in a specialized growth medium (Pricella, CM-0525, Wuhan, China). HFF cells were maintained in DMEM (Gibco, Thermo Fisher Scientific, C11995500BT) supplemented with 10% FBS and 1% Pen-Strep. Primary MEFs were isolated from mouse embryos at embryonic day 13.5, dissociated using trypsin, and cultured in DMEM supplemented with 10% FBS and 1% Pen-Strep. AT2 cells were cultured under three-dimensional conditions as described previously. In addition, HEK-293T cells were cultured in DMEM medium supplemented with 10% FBS. All cell cultures were incubated at 37°C in a humidified atmosphere with 5% CO 2 , and cells were passaged every 2–3 days or upon reaching 70–80% confluence using 0.25% trypsin-EDTA (Gibco, 25200056). For subsequent experiments, cells were dissociated into single-cells suspensions using trypsin-EDTA and seeded into new culture plates at the required cell densities. After overnight incubation to allow for cell attachment, specific treatments or transfections were performed according to established experimental protocols, ensuring optimal conditions and reliable results for further analyses. Cellular Senescence Induction In vitro cultured cells were induced to senescence by treatment with 10 µM Bleomycin (MedChemExpress, HY17565) 18 , 10 µM Nutlin-3a (MedChemExpress, HY10029) 13 , or 200 µM hydrogen peroxide (H 2 O 2 , Sigma, 323381) 29 for six days, with the culture medium refreshed every three days to maintain compound concentration and sufficient nutrients. For senescence induction, cells were seeded in 6-well plates at a density of 1×10⁵ cells/mL one day prior to initiating small molecule treatments, ensuring optimal attachment and consistent growth. Following treatment, senescence induced by each treatment condition was confirmed by staining of SA-β-Gal, p53, p21, and Ki67. Senolytic Agents Evaluation The efficacy and selectivity of several senolytic agents were systematically evaluated in both senescent and non-senescent cells. The agents tested included PES, D&Q, MKT-077 (MedChemExpress, HY15096), ABT-263 (MedChemExpress, HY10087), and BPTES (MedChemExpress, HY12683). Senescence was induced through prolonged passaging, oxidative stress, or drug-induced stress, as described in figure legends. Each compound was applied at optimal concentrations determined through preliminary dose-response cytotoxicity assays. Cells were treated for six days, with culture media refreshed every three days to maintain compound concentration and remove dead cells. The selectivity of senolytic agents was assessed by measuring differences in cell viability between senescent and non-senescent populations, quantified via CellTiter-Lumi assays. For mechanistic studies, additional small molecules were employed individually or in combination with senolytic agents to modulate apoptotic and lysosomal regulation. These included CA-074 (MedChemExpress, HY103350), a selective cathepsin B inhibitor; Cathepsin Inhibitor 1 (MedChemExpress, HY100231), a broad-spectrum cathepsin inhibitor; HSF1A (MedChemExpress, HY103000), an activator of heat shock factor 1 (HSF1); as well as apoptosis inhibitors Z-VAD-FMK (MedChemExpress, HY-16658B) and Q-VD-OPh (MedChemExpress, HY-12305). Cell viability Assay Cell viability was assessed using the CellTiter-Lumi Plus Luminescent Cell Viability Assay Kit (Beyotime Biotechnology, China, C0068), following the manufacturer's recommended protocol. Briefly, cells were seeded into 96-well plates at a density of 5,000 cells per well and incubated overnight to ensure optimal attachment. The following day, cells were exposed to the specific compounds with corresponding concentrations and durations, as detailed in the figure legends. After treatment, cell viability assay reagents were sequentially added to each well according to the manufacturer's instructions. Plates were then incubated for 10 minutes at room temperature to allow for complete reaction, and luminescence was measured using a microplate reader (PerkinElmer EnSpire, USA). All experiments were independently performed at least three times to ensure consistency, reliability, and reproducibility. Data were normalized to untreated control and presented as relative cell survival. To determine the selectivity of each senolytic agent, the survival rate of non-senescent cells (𝑆 non−senescence ) and the survival rate of senescent cells (𝑆 senescence ) were measured after treatment. The ratio of the survival rate of non-senescent cells to senescent cells (R) was defined as: R = \(\:\frac{Snon-senescence}{Ssenescence}\) A higher R value indicates a lower toxicity towards non-senescent cells compared to senescent cells, reflecting greater selectivity. Conversely, a lower R indicates higher toxicity towards non-senescent cells, reflecting poor selectivity. Acridine Orange Staining Cells were seeded into confocal culture dishes (Cellvis, D35C4-20-1-N) at a density of 1×10 4 cells per well and cultured in the appropriate medium. After treatment with the specific compounds for corresponding concentrations and durations, as detailed in the figure legends, the culture medium was gently aspirated and replaced with fresh medium supplemented with acridine orange (MACKLIN, A861550) at a final concentration of 0.1 µg/ml. Subsequently, the cells were incubated at 37°C for 30 minutes to allow for sufficient staining. following incubation, the staining solution was carefully aspirated, and cells were gently washed three times with PBS to remove any excess or unbound dye. Finally, the PBS was replaced with fresh, dye-free culture medium, and high-resolution fluorescence images were acquired using a laser scanning confocal microscope integrated with a live-cell imaging system. Plasmid Construction and Lentiviral Packaging In this study, we constructed lentiviral plasmids engineered to overexpress HspA1A , HspA1A W90F 28 , and HspA1A 2 CA 24 using a doxycycline-controlled expression system. The full-length cDNA encoding HspA1A was procured from Shanghai HeWu Biological Technology Co., Ltd. and precisely integrated into the lentiviral vector by homologous recombination, employing the ClonExpress® Ultra One Step Cloning Kit (Vazyme, Nanjing, China, C115). Point mutations generating the HspA1A W90F and HspA1A 2 CA mutants were introduced via PCR-based site-directed mutagenesis and incorporated into the lentiviral vectors following the same recombination protocol. For lentiviral particle production, the doxycycline-inducible constructs were co-transfected into HEK293T cells alongside the packaging plasmids psPAX2 (Addgene, #12260) and the envelope plasmid pMD2.G (Addgene, #12259). Viral particles were harvested, filtered, and used to transduce target cells. Finally, stable cell lines expressing doxycycline-inducible wild-type HspA1A or its mutants were subsequently established and maintained through rigorous puromycin-based (MedChemExpress, HY-B1743) selection. Real-time PCR Total mRNA was extracted from mouse tissue samples and cultured cells using the AxyPrep™ Multisource Total RNA Miniprep Kit (Axygen, AP-MN-MS-RNA-250), according to the manufacturer's instructions. For tissue samples, specimens underwent multiple freeze-thaw cycles in liquid nitrogen followed by thorough homogenization using a pestle to ensure efficient RNA extraction. The purified RNA samples were reverse-transcribed into complementary DNA (cDNA) utilizing the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen, AT311), which simultaneously eliminates genomic DNA and synthesizes high-quality cDNA. Quantitative PCR (qPCR) assays were conducted with the PerfectStart Green qPCR SuperMix (TransGen, AQ601) following the manufacturer's recommended protocols. Specific primer sequences employed for the qPCR reactions are detailed in Table S1 . Relative mRNA expression levels of target genes were calculated using the 2 −ΔΔCt method with normalization to GAPDH as the housekeeping gene. Histological Fixation and Staining Mouse lung tissues were initially fixed in 4% paraformaldehyde (PFA) to preserve tissue morphology. Tissues were then progressively dehydrated through a graded ethanol series (75%, 85%, 95%, and 100%) and cleared in xylene to facilitate paraffin infiltration. Following embedding in paraffin, tissue blocks were sectioned at a uniform thickness of 5 µm using a precision microtome, and the resulting sections were carefully mounted onto clean, adhesive-coated glass slides for further analysis. For hematoxylin and eosin (H&E) staining, tissue sections were first deparaffinized in xylene, and then rehydrated through decreasing ethanol concentrations, followed by a brief rinse in distilled water. Sections were subsequently stained sequentially with hematoxylin and then eosin to clearly visualize the general tissue architecture. Finally, the stained sections were dehydrated through an ascending ethanol series, cleared in xylene, and permanently mounted with neutral mounting medium. Masson’s trichrome staining was employed to visualize collagen deposition in lung tissues. Following the same deparaffinization and rehydration procedures described above, tissue sections were stained according to standard Masson’s trichrome staining protocols. This method distinctly highlighted collagen fibers, enabling a precise assessment of fibrosis and extracellular matrix remodeling in the tissue samples. For detection of SA-β-Gal activity, mouse tissues were carefully embedded in optimal cutting temperature (OCT) compound, rapidly snap-frozen in liquid nitrogen to preserve enzyme activity, and subsequently stored at -80°C. Frozen sections (~ 8 µm thick) were then cut using a cryostat, mounted onto positively charged slides, and stained using the β-galactosidase staining kit (Beyotime Biotechnology, China, C0602) according to the manufacturer's instructions. All prepared histological slides were examined and captured using a high-resolution optical microscope. Immunofluorescence Staining Cultured cells were gently rinsed three times with PBS and fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature. Following fixation, cells were permeabilized with 0.2% Triton X-100 for 10 minutes to facilitate antibody penetration. To minimize nonspecific binding, cells were blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature. Primary antibodies (diluted as specified in Table S2) were then applied, and cells were incubated overnight at 4°C. The next day, cells were thoroughly washed with PBS and incubated with fluorescent-dye conjugated secondary antibodies at room temperature for 1 hour in the dark. Mouse tissue sections were first deparaffinized by immersion in fresh xylene for 10 minutes, repeated twice. Following deparaffinization, the sections were sequentially rehydrated through a graded ethanol series: two washes in 100% ethanol (5 minutes each), followed by successive washes in 95%, 85%, and 75% ethanol (3 minutes each), and finally rinsed thoroughly in distilled water. Antigen retrieval was performed by immersing the slides in Tris-EDTA antigen retrieval buffer (Solarbio, C1038) and heating in a microwave oven at medium power for 15 minutes. After natural cooling to room temperature, the sections were gently washed three times with PBS (5 minutes each). To reduce nonspecific antibody binding, sections were blocked with 5% bovine serum albumin (BSA) in PBS for 1 hour at room temperature. Primary antibodies, (diluted as specified in Table S2) were then applied, and the sections were incubated overnight at 4°C in a humidified chamber. The following day, sections were thoroughly washed three times with PBS (5 minutes per wash) to remove unbound antibodies, and then incubated with fluorescent-dye conjugated secondary antibodies (as detailed in Table S2) at room temperature for 1 hour in the dark. Finally, after additional PBS washes to remove excess secondary antibody, the stained sections were mounted with antifade mounting medium. High-resolution immunofluorescence images were captured using a Nikon A1R HD25 laser scanning confocal microscope, enabling precise visualization and quantification of protein localization and expression patterns in cell and tissue samples. Western blot analysis Cultured cells and mouse tissue samples were lysed thoroughly using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, China, P0013B) freshly supplemented with Protease and Phosphatase Inhibitor Cocktail (Beyotime Biotechnology, China, P1045) to preserve protein and its phosphorylation. Samples were incubated on ice for 30 minutes with intermittent gentle mixing to ensure complete lysis and extraction. Following incubation, lysates were clarified by centrifugation at 12,000 × g for 15 minutes at 4°C. The supernatants containing the extracted proteins were then carefully collected, and protein concentrations were quantified using the Bicinchoninic Acid (BCA) Protein Assay Kit (Beyotime Biotechnology, China, P0399). For Western blot analysis, equal amounts of protein (typically 20–40 µg per lane) were loaded onto SDS-PAGE gels and separated by electrophoresis. Proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes (Merck, ISEQ00010). Membranes were blocked for 1 hour at room temperature using QuickBlock™ Western Blocking Buffer (Beyotime Biotechnology, China, P0252) to reduce nonspecific antibody binding. After blocking, membranes were incubated overnight at 4°C with specific primary antibodies diluted in QuickBlock™ Western Primary Antibody Dilution Buffer (Beyotime Biotechnology, China, P0256), as detailed in Table S2. The next day, membranes were extensively washed with Tris-buffered saline containing 0.1% Tween-20 (TBST) to remove unbound primary antibodies. They were then incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (listed in Table S2) for 1 hour at room temperature. After additional TBST washes, protein bands were visualized using an enhanced chemiluminescence (ECL) detection system (Yeasen Biotechnology, Shanghai, China, 36208ES76). Images were subsequently captured and analyzed using the Amersham Imager 600 imaging system (GE Healthcare) to ensuring high-resolution and accurate protein quantification. Pulmonary Function Testing Mice under normal physiological conditions were analyzed using a whole-body plethysmography system (WBP, emka Technologies). Each mouse was individually placed into the plethysmograph chamber for a minimum of 10 minutes to allow for acclimation and stabilization of normal respiratory patterns. Following this habituation period, various respiratory parameters were continuously measured and recorded, including Inspiratory Time (Ti), Expiratory Time (Te), Peak Inspiratory Flow (PIF), Peak Expiratory Flow (PEF), Minute Volume (MV), Breathing Frequency (F), and Relaxation Time (RT). To account for differences in body size and metabolic status among the mice, MV was normaizled to body weight (MV/BW), providing a more accurate and physiologically meaningful assessment of pulmonary ventilation efficiency. MicroCT Scanning Lung imaging of mice was performed using a Quantum GX MicroCT imaging system (PerkinElmer, MA, USA) to obtain high-resolution visualization of pulmonary structures. The scanning parameters were carefully optimized to ensure high image quality, including an X-ray voltage of 90 kV, an X-ray current of 88 µA, a field of view (FOV) of 45 mm, and a voxel resolution with a pixel size of 90 µm. Prior to scanning, mice were deeply anesthetized and meticulously positioned on the scanning platform to minimize motion artifacts. Sequential lung image datasets were automatically acquired during the scan, and upon completion, the data were processed for reconstruction and segmentation to generate clear anatomical images of the lung. Quantitative and Statistical analysis ImageJ software (National Institutes of Health, Bethesda, MD, USA) was utilized to quantify mean fluorescence intensities from immunofluorescence images, and to measure the grayscale values of protein bands from western blot analyses. Graphs illustrating these quantitative results were generated using GraphPad Prism software (GraphPad Software, San Diego, CA, USA). Statistical tests were performed using appropriate tests based on experimental design: Student's t-test was applied for pairwise comparisons between two groups, and one-way analysis of variance (ANOVA) followed by Tukey's post hoc test was conducted for comparisons among three or more groups. Data are presented as mean ± standard error of the mean (SEM). Statistical significance was defined as *P < 0.05, **P < 0.01, and ***P < 0.001. All experiments were independently repeated at least three times to ensure robustness, accuracy, and reproducibility of the findings. Declarations Acknowledgements We thank the Imaging Core Facility, Technology Center for Protein Sciences, Tsinghua University for the assistance in using Nikon A1R HD25 laser confocal microscope, and the Laboratory Animal Research Center, Tsinghua University for the assistance in animal experiments. This work is supported by the National Key R&D Program of China (2022YFA1103704 to S.D.), Beijing Natural Science Foundation (JQ22016 to T.H.M.), the New Cornerstone Investigator Program (to S.D.), and Tsinghua-Peking joint Center for Life Sciences (to S.D.). Author contributions Y.L.F., T.H.M and S.D. designed the project, discussed the results, and approved the final version of the manuscript. Y.L.F. performed the experiments and analyzed the data, wrote the manuscript, and supervised the project. F.F.J. and Y.X.Z participated in the animal experiments. Y.L.F., Y.Z., F.F.J., T.H.M and S.D. participated in the discussion of results. S.D. is the lead contact. 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Zhou, Y., et al. Pifithrin-mu is efficacious against non-small cell lung cancer via inhibition of heat shock protein 70. Oncol Rep 37, 313–322 (2017). Zhu, H., et al. Pifithrin-mu incorporated in gold nanoparticle amplifies pro-apoptotic unfolded protein response cascades to potentiate synergistic glioblastoma therapy. Biomaterials 232, 119677 (2020). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryInformation.doc Discovery of Hsp70-Mediated Lysosomal Repair as a Highly Specific Target for Senolysis Cite Share Download PDF Status: Under Review Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-6662488","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":463021110,"identity":"a9ed5edf-7702-478d-96cc-ba52b71dafa4","order_by":0,"name":"Sheng 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09:12:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6662488/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6662488/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83589305,"identity":"d003b677-74b2-431e-ad22-662cc4f78d43","added_by":"auto","created_at":"2025-05-29 05:30:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":812536,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHsp70 inhibition induces significant cell death in SnCs, but not in non-SnCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Molecular structure of PES.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e, Representative images showing the non-senescent (non-SnCs) and senescent (SnCs) A549 cells treated with or without 10 μM PES, Scale bar: 500μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e, Cell viability of non-senescent A549 cells and senescent A549 cells induced by DNA damage (D-SnCs), Nutlin3a (N-SnCs), and oxidative stress (Ox-SnCs), following treatment with the specified concentrations of PES for 6 days. n=5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e, R values presenting differential cell viability between the senescent and non-senescent A549 cells shown in (c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e, Cell viability in senescent A549 cells (D-SnCs, N-SnCs and Ox-SnCs), following knockdown of \u003cem\u003eHspA1A\u003c/em\u003e. n=5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003e, Western blot analysis showing Hsp70 expression in senescent A549 cells (D-SnCs) following treatment with or without doxycycline.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg\u003c/strong\u003e, Cell viability in the same senescent A549 cells ((D-SnCs)) as shown in (f) following treatment with various concentrations of PES. n=3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eh\u003c/strong\u003e, Cell viability in senescent A549 cells (D-SnCs) overexpressing \u003cem\u003eHspA1A\u003c/em\u003e and \u003cem\u003eHspA1A\u003c/em\u003e\u003csup\u003e2CA\u003c/sup\u003e, following treatment with 50 mM PES. n=5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei\u003c/strong\u003e, Cell viability in senescent A549 cells (D-SnCs) and in those pretreated with HSF1A following treatment with the indicated concentrations of PES. n=9.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SEM; *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, and ****P \u0026lt; 0.0001, determined by two-tailed Student’s t test.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6662488/v1/b8c8fef0416f49d64e7a22f1.png"},{"id":83589303,"identity":"76fe903f-96a4-4be1-b801-1280af61355c","added_by":"auto","created_at":"2025-05-29 05:30:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1049093,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHsp70 is enriched in the lysosomal membrane and facilitates membrane repair exclusively in SnCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,\u003c/strong\u003e Representative images showing the co-localization of Hsp70 with Lamp1 in non-senescent A549 cells and senescent A549 cells (D-SnCs, N-SnCs and Ox-SnCs).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e, Representative immunofluorescence images depicting ceramide levels in the non-SnCs and SnCs shown in (a).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e, Quantitative analysis of fluorescence intensity of ceramide in (b). n=10 randomly selected fields.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e, Representative immunofluorescence images showing ceramide levels in senescent A549 cells (D-SnCs) and in D-SnCs overexpressing \u003cem\u003eHspA1A\u003c/em\u003e or \u003cem\u003eHspA1A\u003c/em\u003e\u003csup\u003e2CA\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e, Quantitative analysis of the fluorescence intensity of ceramide in (d). n=15 randomly selected fields.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef, \u003c/strong\u003eRepresentative\u003cstrong\u003e \u003c/strong\u003eimmunofluorescence images displaying Gal8 (upper) and CTSD (lower) staining in the same cells as in (d).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg,\u003c/strong\u003e Quantitative analysis of the number of Gal8 puncta per cell in (f). n=15 randomly selected fields.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eh\u003c/strong\u003e, Quantitative analysis of the mean fluorescence intensity of nuclear CTSD in (f). n=10 randomly selected fields.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei, \u003c/strong\u003eRepresentative fluorescence images showing lysosomal acidity in the same cells as in (d).\u003c/p\u003e\n\u003cp\u003eScale bar: 20 μm. Data are presented as mean ± SEM; *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, and ****P \u0026lt; 0.0001, determined by two-tailed Student’s t test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ej, \u003c/strong\u003eSchematic illustration depicting how Hsp70 binds to BMP, which in turn binds to and activates ASM, leading to the hydrolysis of sphingomyelin and production of ceramide, thereby preventing extensive LMP in senescent cells, CL: Cytosolic leaflet, ILL: Intraluminal leaflet.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6662488/v1/d024ac1fd7dbdb3d17a19b56.png"},{"id":83589584,"identity":"e4d05725-b788-436c-ae05-41ddad2cc1d7","added_by":"auto","created_at":"2025-05-29 05:38:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":850742,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePES induces Catastrophic LMP in SnCs, thereby triggering LDCD.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,\u003c/strong\u003e Representative immunofluorescence images showing the co-localization of Gal3 with lysosomal membrane protein Lamp1 in senescent A549 cells (D-SnCs) following treatment with or without 10 μM PES. The colocalization coefficient of Gal3 with Lamp1 was quantified and indicated at bottom. n=10. Scale bar: 20 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb,c, \u003c/strong\u003eRepresentative images\u003cstrong\u003e \u003c/strong\u003e(b) and quantitative analysis (c) showing the lysosomal acidity in D-SnCs treated with or without 10 μM PES, as indicated by the red fluorescence (R-Fluo) in acridine orange staining. n=11 randomly selected fields. Scale bar: 20 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e, Representative immunofluorescence images showing the altered subcellular distribution of CTSB and CTSD in D-SnCs following treatment with 10 μM PES for 2 days. Scale bar: 20 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e, Representative immunofluorescence images showing the co-localization of doxycycline-inducible exogenous Hsp70\u003cem\u003e \u003c/em\u003ewith lysosomal membrane protein Lamp1 in D-SnCs treated with 10 μM PES. The colocalization coefficient of exogenous Hsp70 with Lamp1 was quantified and indicated at bottom. Scale bar: 20 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef, \u003c/strong\u003eRepresentative immunofluorescence images showing the different subcellular localization of Gal3 between D-SnCs with and without doxycycline-inducible expression of exogenous Hsp70 expression following 10 μM PES treatment. Scale bar: 20 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg, \u003c/strong\u003eQuantitative analysis presenting the ratio of nuclear to cytoplasmic fluorescence intensity of Gal3 in the cells as shown in (f). n=15 randomly selected fields.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eh,\u003c/strong\u003e Quantitative analysis showing the cell viability in D-SnCs in the absence or presence of cathepsin inhibitors (CA-047 and Cathepsin inhibitor 1, CTSi1) following treatment with PES at indicated concentrations. n=4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei\u003c/strong\u003e, Bright field images displaying SnCs pre-treated with or without anti-apoptotic small molecules (Z-VAD-FMK [ZVF, 10 μM] and Q-VD-Oph [QVO, 10 μM]) for 8 hours prior to treatment with or without 10 μM PES. Scale bar: 300 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ej, \u003c/strong\u003eQuantitative analysis showing the cell viability in (i). n=3.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SEM; *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, and ****P \u0026lt; 0.0001, determined by two-tailed Student’s t test.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6662488/v1/d76e7afa1ad9be7e4165adab.png"},{"id":83589579,"identity":"5455b833-b493-4e19-b6e0-5169625f813c","added_by":"auto","created_at":"2025-05-29 05:38:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":737590,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePES and D\u0026amp;Q treatment alleviates aging-related phenotypes in mice undergoing WBI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Schematic representation of the protocol used to evaluate the \u003cem\u003ein vivo\u003c/em\u003e effects of PES and D\u0026amp;Q in mice with WBI-induced accelerated aging.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e, Representative whole-body image showing hair color in the normal (naïve) mice and in mice subjected to WBI, following treatment with vehicle, PES, or D\u0026amp;Q.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec, \u003c/strong\u003eRepresentative images exhibiting the SA-β-Gal staining in the lung tissue of mice shown in (b). Scale bar: 100 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed,\u003c/strong\u003e Quantitative analysis of the SA-β-Gal staining in (c) showing the number of SA-β-Gal-positive cells per mm\u003csup\u003e2 \u003c/sup\u003ewithin the lung tissue aera. n=10.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee,f,g\u003c/strong\u003e, qPCR (e,f) and western blot (g) analysis showing the mRNA levels of \u003cem\u003ep16\u003c/em\u003e\u003csup\u003eINK4A\u003c/sup\u003e(e) and \u003cem\u003ep21\u003c/em\u003e (f), as well as the protein expression of p21 (g) in lung tissues from mice shown in (b). n=12.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eh\u003c/strong\u003e, qPCR analysis showing \u003cem\u003eHspA1A\u003c/em\u003e expression level in the lung tissues of mice as shown in (b). n=12.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei,j\u003c/strong\u003e, Quantitative analysis revealing grip strength (i) (n=6) and treadmill endurance (j) (n=5) in the mice shown in (b).\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SEM; *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, and ****P \u0026lt; 0.0001, determined by two-tailed Student’s t test.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6662488/v1/ed9f60aad1299ea3f8f362fd.png"},{"id":83589302,"identity":"557719a3-a40c-4b68-b9bd-8e8885470d13","added_by":"auto","created_at":"2025-05-29 05:30:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1101655,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePES treatment significantly alleviates pulmonary fibrosis in mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Schematic illustration of the experimental protocol used to evaluate the in vivo effects of PES and D\u0026amp;Q in the mice with pulmonary fibrosis, established by prior treatment with bleomycin and compared to control mice pre-trereated with PBS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e, Western blot analysis displaying the protein levels of senescence markers (p16\u003csup\u003e INK4A\u003c/sup\u003e and p21) in lung tissue of the mice shown in (a).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e, Representative immunofluorescence images showing p16\u003csup\u003eINK4A\u003c/sup\u003e staining in lung tissue from mice as shown in (a). Scale bar: 20μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed,\u003c/strong\u003e Quantitative analysis of the mean fluorescence intensity of p16\u003csup\u003eINK4A\u003c/sup\u003e in (c). n=10.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e, Representative Immunofluorescence images presenting the staining of fibrosis markers (α-SMA, Collagen I, and Fibronectin) in lung tissue from mice as shown in (a). Scale bar: 20μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef-h\u003c/strong\u003e, Quantitative analysis revealing the mean fluorescence intensity of α-SMA (f), Collagen I (g), and Fibronectin (h) in (e). n=10.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei-p\u003c/strong\u003e, Whole-body plethysmography (WBP) analysis of lung function showing Minute Volume (i), Minute Volume normalized to Weight (j), Inspiratory Time (k), Expiratory Time (l), Peak Inspiratory Flow (m), Peak Expiratory Flow (n), Relaxation Time (o), and Breathing Frequency (p) in the mice shown in (a). n=5.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SEM; *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, and ****P \u0026lt; 0.0001, determined by two-tailed Student’s t test.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6662488/v1/f2e10d6c87a705169772cab4.png"},{"id":85388570,"identity":"215b05dc-6ce9-4a55-b2db-75e64c7cc68f","added_by":"auto","created_at":"2025-06-25 10:09:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5385480,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6662488/v1/90a5feb0-3ccf-4b4e-bd1d-784ef03b84e2.pdf"},{"id":83589308,"identity":"1b0201af-1848-41fd-99cf-c6cbe154f243","added_by":"auto","created_at":"2025-05-29 05:30:27","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14309888,"visible":true,"origin":"","legend":"Discovery of Hsp70-Mediated Lysosomal Repair as a Highly Specific Target for Senolysis","description":"","filename":"SupplementaryInformation.doc","url":"https://assets-eu.researchsquare.com/files/rs-6662488/v1/2e63dbcaa27c7c7eda642bb3.doc"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Discovery of Hsp70-Mediated Lysosomal Repair as a Highly Specific Target for Senolysis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSnCs are significant contributors to aging and a wide range of age-related diseases, including tissue fibrosis, osteoarthritis, Alzheimer\u0026rsquo;s disease, and cancer\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Cellular senescence can be induced by DNA damage, oncogene activation, or excessive cellular replication\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Once senescent, these cells evade apoptosis and secrete pro-inflammatory factors\u0026mdash;collectively referred to as the senescence-associated secretory phenotype (SASP) factors\u0026mdash;which contribute to tissue dysfunction and accelerate aging and age-related disease progression\u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Consequently, targeted elimination of SnCs (senolysis) has emerged as a promising therapeutic strategy for tissue rejuvenation and the treatment of age-related disorders\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15 CR16 CR17 CR18\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSeveral senolytic compounds, including Navitoclax (ABT-263), Fisetin, and the Dasatinib-Quercetin (D\u0026amp;Q) combination, have shown efficacy in clearing SnCs both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. However, their clinical application is limited by poor selectivity and significant adverse effects, largely because they target anti-apoptotic pathways that are also critical for the survival of normal cells\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Additionally, the inherent heterogeneity of SnCs, which is characterized by diverse phenotypes and molecular profiles, further complicates the development of broadly effective senolytic strategies\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Therefore, identifying vulnerabilities that are specific to SnCs and absent in non-SnCs may provide a path toward more selective and effective senolytic approaches.\u003c/p\u003e \u003cp\u003eIn SnCs, lysosomes have been shown to exhibit reduced acidity and compromised membrane integrity\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. However, identifying a senescent cell-specific mechanism of lysosomal modulation and directly targeting such lysosomal vulnerability in a senescent cell-specific manner remains underexplored. In this study, we serendipitously discovered that PES\u003csup\u003e\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, through inhibition of Hsp70, exhibits unprecedented selective senolytic activity by inducing significant cell death exclusively in SnCs while sparing non-SnCs. Mechanistic investigations revealed a previously unrecognized role for Hsp70 in repairing defective lysosomal membranes to maintain SnC survival. This finding is consistent with Hsp70\u0026rsquo;s role in stabilizing lysosomes in Niemann\u0026ndash;Pick disease, where mutations in the acid sphingomyelinase (ASM) gene result in markedly reduced lysosomal stability\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Inhibition of Hsp70 by PES disrupts this repair mechanism, inducing extensive LMP and cathepsins release-triggered LDCD, ultimately leading to the selective elimination of SnCs. Moreover, in vivo treatment with PES selectively cleared SnCs, and consequently ameliorated aging phenotypes and reversed lung fibrosis in mouse models of irradiation-induced accelerated aging and pulmonary fibrosis, respectively. These findings establish Hsp70 inhibition as a novel, highly selective, and broadly effective senolytic strategy that targets the lysosomal membrane repair mechanism specifically activated in SnCs, thereby offering a promising therapeutic approach for treating aging and age-related diseases.\u003c/p\u003e\n\u003ch3\u003ePES induces selective senolysis via Hsp70 inhibition\u003c/h3\u003e\n\u003cp\u003eIn our investigation of SnCs, we unexpectedly discovered that treatment with a small molecule, PES, induced significant cell death in senescent A549 cells (an adenocarcinomic human alveolar basal epithelial cell line) while largely sparing non-SnCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea,b). To assess whether PES-mediated senolysis is broadly effective across different cell types with senescence induced by distinct mechanisms, we tested its senolytic activity across multiple cell lines, including H1299 (a human non-small cell lung carcinoma cell line, p53-null), MCF10A (a non-tumorigenic human mammary epithelial cell line), and HFF1 (a normal human foreskin fibroblast cell line). Senescence was induced under various conditions: Bleomycin-induced DNA damage (D-SnCs)\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, Nutlin-3a-induced p53 activation (N-SnCs)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress (Ox-SnCs)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea,b). Notably, treatment with 10 \u0026micro;M PES induced cell death in over 50% of SnCs in all tested cell lines while exerting negligible effects on their corresponding non-SnCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, Fig.S2a-c). Similarly, PES induced selective senolysis in replication-induced senescent mouse embryonic fibroblasts (R-SnCs) compared to their non-senescent counterparts (Fig.S2d). To compare the selectivity of PES-mediated senolysis with that of previously identified senolytic agents, we calculated their selectivity by determining the viability ratio (R), which is the percentage of viable non-SnCs relative to SnCs at the same treatment concentration. Our analysis revealed that PES exhibits selectivity several orders of magnitude greater than previously identified senolytic agents, such as ABT-263, D\u0026amp;Q, and BPTES (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, Fig.S3a-c). Collectively, these results demonstrate that PES induces senolysis with significantly greater selectivity than other senolytic agents, suggesting that its targeting mechanism is crucial for SnC survival but not for non-SnCs.\u003c/p\u003e \u003cp\u003eIt has been previously established that PES inhibits Hsp70. To confirm that PES induces selective senolysis through Hsp70 inhibition, we compared its effect with another Hsp70 inhibitor, MKT-077\u003csup\u003e30\u003c/sup\u003e, on SnCs and non-SnCs. Consistent with the PES findings, SnCs exhibited significant cell death upon MKT-077 treatment (Fig.S4a), while non-SnCs remained largely viable. To further confirm the specificity of Hsp70 targeting, shRNA-mediated knockdown of \u003cem\u003eHspA1A\u003c/em\u003e (a specific gene that encodes Hsp70 protein) induced selective senolysis, resulting in approximately 50% cell death in SnCs while leaving non-SnCs nearly unaffected (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Next, we established a doxycycline-inducible system in SnCs to overexpress either wild-type \u003cem\u003eHspA1A\u003c/em\u003e or its mutant form, \u003cem\u003eHspA1A\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003eCA\u003c/sup\u003e, which prevents PES binding by cysteine-to-alanine mutations at positions 574 and 603\u003csup\u003e24\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Consistent with the expected mechanism of rescuing target inhibition, overexpression of wild-type \u003cem\u003eHspA1A\u003c/em\u003e significantly protected SnCs from PES- or MKT-077-induced cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, Fig.S4b). Furthermore, SnCs overexpressing the PES-resistant mutant \u003cem\u003eHspA1A\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003eCA\u003c/sup\u003e exhibited greater resistance to PES-induced cell death, with over 80% survival at 50 \u0026micro;M PES\u0026mdash;a concentration that induced nearly complete cell death in cells overexpressing wild-type \u003cem\u003eHspA1A\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). Moreover, a small-molecule activator of Heat Shock Transcription Factor 1 (HSF1), HSF1A, which enhances endogenous \u003cem\u003eHspA1A\u003c/em\u003e transcription, similarly rescued PES- or MKT-077-induced cell death in SnCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei, Fig.S4c). Collectively, these findings confirmed that PES induces selective senolysis through Hsp70 inhibition, leading to significant cell death in SnCs while sparing non-SnCs.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHsp70 Facilitates Lysosomal Membrane Repair in SnCs\u003c/h2\u003e \u003cp\u003eTo elucidate the mechanism by which Hsp70 specifically maintains the survival of SnCs and how this is disrupted by PES, we first examined whether Hsp70 is differentially expressed between SnCs and non-SnCs. qPCR and Western blot analyses revealed that both mRNA and protein levels of Hsp70 were significantly higher in SnCs than in non-SnCs, which was consistent with more pronounced nuclear accumulation of HSF1 in SnCs (Fig.S5a-d). To further characterize Hsp70 subcellular localization, immunostaining revealed that Hsp70 was prominently co-localized with the lysosomal-associated membrane protein 1 (Lamp1), a marker of lysosomes, in SnCs, a feature not observed in non-SnCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). This exclusive lysosomal localization suggests that Hsp70 may play a critical role in lysosomal regulation, a mechanism essential for SnC survival. To explore this hypothesis, we characterized lysosomes in SnCs and non-SnCs. Compared to non-SnCs, SnCs exhibited evident nuclear translocation of TFEB\u0026mdash;a master transcription factor driving lysosomal biogenesis\u0026mdash;along with a significant increase in the protein expression of Lamp1 and Lamp2A and lysosomal proteases cathepsin B (CTSB) and cathepsin D (CTSD) (Fig.S6a-d). These results indicate that lysosomal biogenesis is enhanced in SnCs compared to non-SnCs. Additionally, SnCs exhibited mild lysosomal membrane disruption, as evidenced by emergence of Galectin-8 (Gal8, a marker of early lysosomal rupture) puncta, reduced acridine orange staining (indicative of decreased lysosomal acidity), and leakage of CTSD into the endonuclear compartment (Fig.S6e-h). Collectively, these findings highlight the accumulation of lysosomes with mildly disrupted membrane integrity as a unique feature of SnCs, unequivocally distinguishing them from non-SnCs.\u003c/p\u003e \u003cp\u003eGiven that Hsp70 has been previously shown to stabilize lysosomal membrane, particularly in the cells from patients with Niemann\u0026ndash;Pick disease\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, its enrichment at lysosomal membranes in SnCs suggests that it may play a key role in repairing lysosomal membrane in these cells. Consistent with this hypothesis, ASM\u0026mdash;a key enzyme recruited to the defective lysosomal membrane by Hsp70 in acidic environment and catalyzing hydrolyzation of bis(monoacylglycero)phosphate (BMP) into ceramide, an essential lipid in membrane repair\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e\u0026mdash;was found to be highly expressed in SnCs compared to non-SnCs (Fig.S7a). Furthermore, ASM exhibited a lysosomal enrichment pattern similar to that of Hsp70 in SnCs, as evidenced by its co-localization with the Lamp1 (Fig.S7b). In line with these observations, intracellular ceramide levels were markedly higher in SnCs relative to non-SnCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb,c). To assess whether Hsp70 mediates lysosomal membrane repair in an BMP-dependent manner in SnCs, we compared the effects of \u003cem\u003eHspA1A\u003c/em\u003e overexpression in SnCs with that of \u003cem\u003eHspA1A\u003c/em\u003e\u003csup\u003eW90F\u003c/sup\u003e\u0026mdash;a mutant containing a Trp90Phe substitution that specifically abolishes the interaction between Hsp70 and BMP without disrupting its chaperone activity\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Overexpression of \u003cem\u003eHspA1A\u003c/em\u003e or upregulation of its endogenous expression via HSF1A treatment further enhanced ceramide production in SnCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed,e, Fig.S8a,b), whereas overexpression of H\u003cem\u003espA1A\u003c/em\u003e\u003csup\u003eW90F\u003c/sup\u003e failed to elevate ceramide levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed,e). Moreover, \u003cem\u003eHspA1A\u003c/em\u003e overexpression or HSF1A treatment significantly mitigated lysosomal permeability, as indicated by a marked decrease in both Gal8 puncta and endonuclear mislocalization of CTSD, whereas \u003cem\u003eHspA1A\u003c/em\u003e\u003csup\u003eW90F\u003c/sup\u003e overexpression had no such rescuing effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef-h, Fig.S8c,d). Consistently, LysoTracker or acridine orange staining revealed that \u003cem\u003eHspA1A\u003c/em\u003e overexpression or HSF1A treatment enhanced lysosomal acidification in SnCs\u0026mdash;an effect not achieved by overexpressing \u003cem\u003eHspA1A\u003c/em\u003e\u003csup\u003eW90F\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei, Fig.S8e,f). Collectively, these findings demonstrate that Hsp70 is specifically upregulated and recruited to lysosomal membranes in SnCs, but not in non-SnCs, where it facilitates membrane repair by promoting ASM-dependent ceramide production (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePES Selectively induces Extensive LMP to Trigger LDCD in SnCs\u003c/h3\u003e\n\u003cp\u003eGiven our new findings that upregulating and recruiting Hsp70 to lysosome in response to senescence induction can facilitate lysosomal membrane repair in SnCs, we next investigated whether Hsp70 is essential for maintaining lysosomal membrane in these cells, thereby preventing lysosomal membrane disruption to a level that could otherwise compromise cell survival. To test this, we assessed LMP in SnCs following PES treatment using Galectin-3 (Gal3) staining. Gal3, a cytosolic β-galactoside-binding lectin, is recruited to damaged lysosomes and coordinates autophagic responses to irreversible lysosomal injury; its lysosomal localization is a widely accepted marker of severe lysosomal membrane damage\u003csup\u003e\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. In PES-treated SnCs, we observed a marked cytosolic translocation of Gal3, which was co-localized with Lamp1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), indicating severe lysosomal membrane disruption. Supporting this, PES treatment also led to a significant reduction in acridine orange staining, which reflects a decrease in lysosomal acidity, along with a sharp increase in the cytosolic mislocalization of lysosomal CTSB and CTSD (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-d). Notably, these PES-induced changes were observed exclusively in SnCs and not in non-SnCs (Fig.S9a,b), demonstrating that PES selectively induces extensive LMP in SnCs. When overexpressing \u003cem\u003eHspA1A\u003c/em\u003e in SnCs, the prominent lysosomal localization of Hsp70 remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Nuclear disruption, a characteristic downstream consequence of catastrophic LMP, caused by cathepsin release and subsequent nuclear injury\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, was evident in PES-treated SnCs but was almost completely prevented in \u003cem\u003eHspA1A\u003c/em\u003e-overexpressing SnCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). In contrast to the lysosomal translocation of Gal3 in PES-treated SnCs, \u003cem\u003eHspA1A\u003c/em\u003e overexpression effectively blocked this translocation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef,g), confirming that PES induces extensive LMP through Hsp70 inhibition. Since severe leakage of lysosomal contents, particularly cathepsins, triggers LDCD, we hypothesized that PES-induced senolysis is driven by LDCD. Consistent with this hypothesis, PES-induced cell death in SnCs was largely prevented by pharmacological inhibition of cathepsins, which are known to block LDCD, but not by apoptosis inhibitors such as ZVF and QVO (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh-j). Collectively, these findings demonstrate that Hsp70 plays a crucial role in SnCs to prevent extensive LMP-induced LDCD, a protective mechanism for SnC survival that is effectively targeted by Hsp70 inhibitors like PES to induce highly selective senolysis.\u003c/p\u003e\n\u003ch3\u003ePES Mitigates Accelerated Aging in Irradiated Mice\u003c/h3\u003e\n\u003cp\u003eTo evaluate the in vivo effects of PES-induced selective senolysis on aging, we established an aging mouse model using whole-body irradiation (WBI) at a sublethal dose\u0026mdash;a well-established method to accelerate aging in mice, which has been previously used to assess the anti-aging effects of other senolytics\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. One month after exposure to 5 Gy WBI, mice were treated with either PES or the D\u0026amp;Q combination for an additional month, followed by a two-month observation period (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Four months post-irradiation, mice developed noticeably greyed fur, a hallmark sign of aging. Notably, PES treatment reversed this change, restoring hair color to nearly that of non-irradiated (na\u0026iuml;ve) mice, while D\u0026amp;Q treatment resulted in minimal recovery (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Further analysis of lung tissues revealed that WBI significantly increased the number of senescence-associated β-galactosidase (SA-β-Gal)-positive cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec,d), consistent with previous reports showing similar changes\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. In contrast, both PES and D\u0026amp;Q treatments cleared most of the SA-β-Gal\u0026ndash;positive cells within the lungs, reducing their numbers to levels comparable to those seen in na\u0026iuml;ve mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec,d). In line with these findings, the expression of classical senescence markers\u0026mdash;such as p16\u003csup\u003eINK\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003eA\u003c/sup\u003e, p21, and SASP components\u0026mdash;was significantly elevated in the lungs following WBI, but all markers returned to near-baseline levels after treatment with PES or D\u0026amp;Q (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee-g, Fig. S10). Additionally, \u003cem\u003eHspA1A\u003c/em\u003e expression in lung tissues increased approximately sevenfold following WBI, consistent with our \u003cem\u003ein vitro\u003c/em\u003e findings that SnCs upregulate Hsp70 to facilitate lysosomal repair and maintain survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh, Fig.S5c,d). In contrast, treatment with PES or D\u0026amp;Q restored \u003cem\u003eHspA1A\u003c/em\u003e expression to levels similar to those observed in na\u0026iuml;ve mice, confirming their robust efficacy in clearing senescent cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). Functional assessments further showed that PES treatment markedly improved grip strength and treadmill endurance, which had regressed following WBI, exhibiting superior effects compared to D\u0026amp;Q (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei,j). Overall, our findings indicate that PES enables effective clearance of SnCs in vivo, particularly in lung tissue, and significantly surpasses D\u0026amp;Q in mitigating aging-related physiological decline in WBI-accelerated aging mice. This superior efficacy, likely arising from PES\u0026rsquo;s far higher selectivity for SnCs compared to previously identified senolytic agents, underscores the therapeutic potential of pharmacological Hsp70 inhibition for delaying aging and treating age-related diseases.\u003c/p\u003e\n\u003ch3\u003ePES Ameliorates Pulmonary Fibrosis in Mice\u003c/h3\u003e\n\u003cp\u003ePulmonary fibrosis often results from the senescence and exhaustion of alveolar type 2 (AT2) stem cells, which regenerate alveolar type 1 (AT1) cells after lung injury\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Senescent AT2 cells promote fibroblast infiltration and matrix deposition, impairing gas exchange. Although D\u0026amp;Q have shown promise in alleviating these conditions\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, their clinical application is limited by inevitable toxicity due to poor selectivity. PES, a highly selective Hsp70 inhibitor that efficiently clears senescent cells with far greater specificity than existing agents, may provide a safer and more effective senolytic approach for treating pulmonary fibrosis. To test this hypothesis, we first compared the effects of PES and D\u0026amp;Q on bleomycin-induced senescent and non-senescent AT2 cells \u003cem\u003ein vitro\u003c/em\u003e. As expected, D\u0026amp;Q exhibited significant toxicity toward non-senescent AT2 cells (Fig. S11). By contrast, PES efficiently eliminated senescent AT2 cells while having negligible impact on the survival of non-senescent AT2 cells, even at high concentrations such as 30 \u0026micro;M (Fig. S11). These findings highlight the superior selectivity of PES-induced senolysis, with no observable toxicity to normal AT2 cells, compared to previously identified senolytic agent D\u0026amp;Q.\u003c/p\u003e \u003cp\u003eNext, we established a mouse model of pulmonary fibrosis by administering bleomycin to C57BL/6 mice, inducing senescence in AT2 cells, followed by four consecutive weeks of treatment with either PES or D\u0026amp;Q (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Both PES and D\u0026amp;Q treatments significantly reduced the expression of senescence markers\u0026mdash;including p16\u003csup\u003eINK\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003eA\u003c/sup\u003e, p21, and SASP components\u0026mdash;in lung tissue to levels nearly identical to those observed in normal mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, Fig. S12a-g). Immunostaining further confirmed that both treatments substantially decreased the number of p16\u003csup\u003eINK\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003eA\u003c/sup\u003e-positive cells in bleomycin-treated lung tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, d), bringing them to levels comparable to those seen in normal mice. These results consistently indicate that PES effectively eliminates bleomycin-induced SnCs in the lungs. Pulmonary fibrosis progresses when SASP factors secreted from senescent AT2 cells activate fibroblasts and promote their differentiation into myofibroblasts, leading to the increased synthesis and excessive accumulation of collagen and other extracellular matrix (ECM) components (e.g., α-SMA, fibronectin, and MMPs)\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Immunostaining and qPCR analyses revealed that both PES and D\u0026amp;Q treatments significantly reduced the expression and extracellular accumulation of pivotal ECM components, including collagen I, α-SMA, fibronectin, \u003cem\u003eMMP\u003c/em\u003e10, and \u003cem\u003eMMP\u003c/em\u003e12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee-h, Fig. S13). Compared to D\u0026amp;Q, PES was able to reduce extracellular α-SMA and fibronectin accumulation to lower levels and uniquely downregulate \u003cem\u003eMMP\u003c/em\u003e10 and \u003cem\u003eMMP\u003c/em\u003e12 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee-h, Fig. S13). Consistently, CT scans demonstrated that treatment with either agent reduced fibrotic (white) regions in the lungs, indicating a substantial decrease in lung inflammation (Fig. S14). Furthermore, pulmonary fibrosis in bleomycin-treated mice was markedly ameliorated following PES or D\u0026amp;Q treatment, as evidenced by improved alveolar structure and reduced areas of lung parenchymal consolidation (Fig. S15a,b).\u003c/p\u003e \u003cp\u003e Importantly, whole-body plethysmography (WBP) demonstrated that PES treatment fully restored bleomycin-impaired pulmonary function to near-normal levels, as indicated by significant improvements in minute volume normalized to body weight (an index of ventilatory efficiency diminished in severe fibrosis), inspiratory and expiratory times (prolonged when lung compliance is reduced), peak flow rates (suppressed in fibrotic lungs), relaxation time (prolonged due to impaired elastic recoil in severe fibrosis), and breathing frequency (reduced in advanced pulmonary fibrosis) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei-p). In contrast, D\u0026amp;Q treatment only partially improved lung function and did not significantly affect inspiratory and expiratory times, relaxation time, or breathing frequency, reflecting persistent impairment in respiratory cycle, ventilatory efficiency, lung compliance, airway patency, and respiratory rhythm\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei-p). Collectively, these findings demonstrate that PES effectively clears SnCs in the lungs and significantly mitigates pulmonary fibrosis, restoring multiple key aspects of pulmonary function to near-normal levels. Notably, PES outperformed D\u0026amp;Q as a senolytic treatment for pulmonary fibrosis, as evidenced by its superior capacity to exhibit extremely low toxicity to non-senescent AT2 cells and comprehensively improve lung function. Our results further highlight the therapeutic potential of pharmacological Hsp70 inhibition for treating not only pulmonary fibrosis but also a broad range of age-related diseases.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSnCs commonly display lysosomes that are increased in number and size, along with elevated SA-β-Gal activity\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. In these cells, p16\u003csup\u003eINK\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003ea\u003c/sup\u003e promotes the nuclear translocation of TFEB by inhibiting CDK4, which upregulates genes involved in lysosomal biogenesis and increases lysosomal quantity\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. However, despite enhanced lysosomal biogenesis, SnCs also exhibit widespread lysosomal leakage, leading to elevated intralysosomal pH and diminishing hydrolase activity\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. This compromised membrane integrity allows the leakage of lysosomal contents, such as CTSB and CTSD, into the cytoplasm\u0026mdash;and even into the nucleus\u0026mdash; as observed in senescent microglial cells\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e and in our senescent cell models. To mitigate the detrimental effects of severe LMP\u0026mdash;including cathepsins release, uncontrolled degradation of cytosolic proteins, and LDCD\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e\u0026mdash;SnCs likely activate intrinsic lysosomal repair mechanisms. Although several repair pathways, including Hsp70- and ESCRT-mediated lysosomal membrane repair, have been described in various cell types under specific conditions\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, the mechanisms underlying lysosomal membrane repair in SnCs remain largely unexplored. In our study, we found that Hsp70\u0026mdash;a chaperone that facilitates protein folding and prevents the formation of aberrant protein aggregates\u0026mdash;was markedly upregulated and recruited to lysosomal membranes in SnCs, but not in non-SnCs. This selective localization confers Hsp70 with a senescence\u0026ndash;specific role: patching damaged lysosomes and preventing catastrophic LMP and downstream LDCD. These mechanistic insights explain the highly selective elimination of SnCs by PES, which targets Hsp70 and spares non-SnCs. Moreover, our findings suggest that the senolytic selectivity of pharmacological Hsp70 inhibitors could be further enhanced through chemical modifications that promote lysosomal enrichment. Such modifications likely minimize the impact on lysosome-independent Hsp70 functions in non-SnCs, improving efficacy while reducing side effects. Notably, we also uncovered a previously unrecognized pathway through which Hsp70 specifically supports the survival of SnCs, distinct from its known roles in other cell types including cancer cells\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs aging progresses, the accumulation of SnCs contributes to the increased release of SASP components, creating an inflammatory niche that impairs the regenerative capacity of tissue-resident stem cells and drives the development of age-related diseases\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Pulmonary fibrosis\u0026mdash;a progressive and currently incurable lung disease characterized by irreversible scarring and reduced life expectancy\u0026mdash;is primarily driven by the senescence of AT2 cells. Recent studies have shown that senolytic agents such as D\u0026amp;Q and ABT-263 can alleviate pulmonary fibrosis in mouse models, but their safety concerns persist due to the effects on apoptotic regulation in non-SnCs\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. In our study, PES demonstrated significantly higher senolytic selectivity compared to previously identified agents, leading to superior improvements in lung function and fibrosis in a pulmonary fibrosis mouse model. Crucially, PES exhibited negligible toxicity toward non-senescent AT2 cells within a defined concentration range, consistent with preclinical findings showing that PES effectively inhibits cancer cell growth without adverse effects on vital organs\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. These findings not only reveal an unprecedentedly high level of senolytic selectivity for PES but also suggest a promising therapeutic approach for rejuvenation and the treatment of age-related conditions.\u003c/p\u003e "},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eAnimal models\u003c/h2\u003e \u003cp\u003e All animal experiments were conducted in compliance with protocols approved by the Institutional Animal Care and Use Committee of Tsinghua University, Beijing, China. Male C57BL/6 mice used in this study were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were bred and maintained in individually ventilated cages (six mice per cage) under specific-pathogen-free conditions in a controlled environment (temperature: 20\u0026ndash;26\u0026deg;C; humidity: 40\u0026ndash;60%; 12-hour light/12-hour dark cycle).\u003c/p\u003e \u003cp\u003eFor the bleomycin-induced idiopathic pulmonary fibrosis experiments, 6\u0026ndash;8 week-old mice were anesthetized via inhalation of isoflurane and then received an intranasal administration of 5 mg/kg Bleomycin sulfate (Selleck, S1214) or PBS (phosphate-buffered saline) as a control. The mice were monitored daily, and four weeks post-administration, pulmonary fibrosis developed. The animals were then randomized to initiate treatment. Specifically, dasatinib (5 mg/kg) (MedChemExpress, HY10181) plus quercetin (50 mg/kg) (MedChemExpress, cat.no. HY18085) (D\u0026amp;Q) were administrated by oral gavage every four days for four weeks\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, and PES (10 mg/kg) (MedChemExpress, HY10940) was administrated by intraperitoneal injection every two days for four weeks\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. In parallel, treatment with vehicle served as a control. After the treatment period, the mice were monitored for an additional four weeks to allow sufficient time for lung lesion repair. At the study's endpoint, pulmonary function tests and micro-CT imaging were performed prior to euthanizing the mice. For molecular and pathological evaluation, lung tissues were collected for RNA and protein extraction. Additionally, tissues were paraffin-embedded for immunofluorescence analysis, H\u0026amp;E (haematoxylin and eosin) staining, and Masson's staining, or frozen in OCT solution for SA-β-Gal staining.\u003c/p\u003e \u003cp\u003eFor the WBI assay, C57BL/6J mice aged 10\u0026ndash;12 weeks were exposed to a sublethal dose of 5 Gy using an X-Ray irradiator (RS 2000 Pro, RadSource, USA). One month post-irradiation, the mice were randomly assigned to receive treatments with dasatinib plus quercetin and PES for SnC clearance, following the dosing regimen described in the bleomycin-induced IPF model. Physical functions were evaluated by measuring Grip Strength and Endurance. Grip Strength was assessed using a Grip Strength Meter, with final values averaged over five trials, while endurance was measured using a Treadmill (model 47300, Ugo Basile). Prior to testing, the mice underwent a one-week treadmill training period to acclimate to the exercise protocol.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eCell Culture and Maintenance\u003c/h3\u003e\n\u003cp\u003eMultiple cell lines were used in this study, including A549 (human lung carcinoma), H1299 (human non-small cell lung carcinoma), MCF10A (human mammary epithelial), and HFF1 (human foreskin fibroblast), as well as primary mouse embryonic fibroblasts (MEFs) and primary alveolar type 2 (AT2) cells isolated from mouse lung tissue. All cell lines were obtained from reputable suppliers such as the American Type Culture Collection (ATCC, USA). A549 and H1299 cells were cultured in DMEM/F-12(1:1) medium (Gibco, Thermo Fisher Scientific, C11330500BT) supplemented with 10% fetal bovine serum (FBS, Gibco, 16000-044) and 1% penicillin-streptomycin (Pen-Strep, Gibco, 15140-122). MCF10A cells were maintained in a specialized growth medium (Pricella, CM-0525, Wuhan, China). HFF cells were maintained in DMEM (Gibco, Thermo Fisher Scientific, C11995500BT) supplemented with 10% FBS and 1% Pen-Strep. Primary MEFs were isolated from mouse embryos at embryonic day 13.5, dissociated using trypsin, and cultured in DMEM supplemented with 10% FBS and 1% Pen-Strep. AT2 cells were cultured under three-dimensional conditions as described previously. In addition, HEK-293T cells were cultured in DMEM medium supplemented with 10% FBS. All cell cultures were incubated at 37\u0026deg;C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e, and cells were passaged every 2\u0026ndash;3 days or upon reaching 70\u0026ndash;80% confluence using 0.25% trypsin-EDTA (Gibco, 25200056). For subsequent experiments, cells were dissociated into single-cells suspensions using trypsin-EDTA and seeded into new culture plates at the required cell densities. After overnight incubation to allow for cell attachment, specific treatments or transfections were performed according to established experimental protocols, ensuring optimal conditions and reliable results for further analyses.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCellular Senescence Induction\u003c/h2\u003e \u003cp\u003eIn vitro cultured cells were induced to senescence by treatment with 10 \u0026micro;M Bleomycin (MedChemExpress, HY17565)\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, 10 \u0026micro;M Nutlin-3a (MedChemExpress, HY10029)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, or 200 \u0026micro;M hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, Sigma, 323381)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e for six days, with the culture medium refreshed every three days to maintain compound concentration and sufficient nutrients. For senescence induction, cells were seeded in 6-well plates at a density of 1\u0026times;10⁵ cells/mL one day prior to initiating small molecule treatments, ensuring optimal attachment and consistent growth. Following treatment, senescence induced by each treatment condition was confirmed by staining of SA-β-Gal, p53, p21, and Ki67.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSenolytic Agents Evaluation\u003c/h2\u003e \u003cp\u003eThe efficacy and selectivity of several senolytic agents were systematically evaluated in both senescent and non-senescent cells. The agents tested included PES, D\u0026amp;Q, MKT-077 (MedChemExpress, HY15096), ABT-263 (MedChemExpress, HY10087), and BPTES (MedChemExpress, HY12683). Senescence was induced through prolonged passaging, oxidative stress, or drug-induced stress, as described in figure legends. Each compound was applied at optimal concentrations determined through preliminary dose-response cytotoxicity assays. Cells were treated for six days, with culture media refreshed every three days to maintain compound concentration and remove dead cells. The selectivity of senolytic agents was assessed by measuring differences in cell viability between senescent and non-senescent populations, quantified via CellTiter-Lumi assays. For mechanistic studies, additional small molecules were employed individually or in combination with senolytic agents to modulate apoptotic and lysosomal regulation. These included CA-074 (MedChemExpress, HY103350), a selective cathepsin B inhibitor; Cathepsin Inhibitor 1 (MedChemExpress, HY100231), a broad-spectrum cathepsin inhibitor; HSF1A (MedChemExpress, HY103000), an activator of heat shock factor 1 (HSF1); as well as apoptosis inhibitors Z-VAD-FMK (MedChemExpress, HY-16658B) and Q-VD-OPh (MedChemExpress, HY-12305).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCell viability Assay\u003c/h2\u003e \u003cp\u003eCell viability was assessed using the CellTiter-Lumi Plus Luminescent Cell Viability Assay Kit (Beyotime Biotechnology, China, C0068), following the manufacturer's recommended protocol. Briefly, cells were seeded into 96-well plates at a density of 5,000 cells per well and incubated overnight to ensure optimal attachment. The following day, cells were exposed to the specific compounds with corresponding concentrations and durations, as detailed in the figure legends. After treatment, cell viability assay reagents were sequentially added to each well according to the manufacturer's instructions. Plates were then incubated for 10 minutes at room temperature to allow for complete reaction, and luminescence was measured using a microplate reader (PerkinElmer EnSpire, USA). All experiments were independently performed at least three times to ensure consistency, reliability, and reproducibility. Data were normalized to untreated control and presented as relative cell survival.\u003c/p\u003e \u003cp\u003eTo determine the selectivity of each senolytic agent, the survival rate of non-senescent cells (\u0026#119878;\u003csub\u003enon\u0026minus;senescence\u003c/sub\u003e) and the survival rate of senescent cells (\u0026#119878;\u003csub\u003esenescence\u003c/sub\u003e) were measured after treatment. The ratio of the survival rate of non-senescent cells to senescent cells (R) was defined as:\u003c/p\u003e \u003cp\u003e \u003cem\u003eR\u003c/em\u003e=\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{Snon-senescence}{Ssenescence}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003eA higher \u003cem\u003eR\u003c/em\u003e value indicates a lower toxicity towards non-senescent cells compared to senescent cells, reflecting greater selectivity. Conversely, a lower \u003cem\u003eR\u003c/em\u003e indicates higher toxicity towards non-senescent cells, reflecting poor selectivity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAcridine Orange Staining\u003c/h2\u003e \u003cp\u003eCells were seeded into confocal culture dishes (Cellvis, D35C4-20-1-N) at a density of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well and cultured in the appropriate medium. After treatment with the specific compounds for corresponding concentrations and durations, as detailed in the figure legends, the culture medium was gently aspirated and replaced with fresh medium supplemented with acridine orange (MACKLIN, A861550) at a final concentration of 0.1 \u0026micro;g/ml. Subsequently, the cells were incubated at 37\u0026deg;C for 30 minutes to allow for sufficient staining. following incubation, the staining solution was carefully aspirated, and cells were gently washed three times with PBS to remove any excess or unbound dye. Finally, the PBS was replaced with fresh, dye-free culture medium, and high-resolution fluorescence images were acquired using a laser scanning confocal microscope integrated with a live-cell imaging system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid Construction and Lentiviral Packaging\u003c/h2\u003e \u003cp\u003eIn this study, we constructed lentiviral plasmids engineered to overexpress \u003cem\u003eHspA1A\u003c/em\u003e, \u003cem\u003eHspA1A\u003c/em\u003e\u003csup\u003eW90F \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003eHspA1A\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003eCA \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e using a doxycycline-controlled expression system. The full-length cDNA encoding \u003cem\u003eHspA1A\u003c/em\u003e was procured from Shanghai HeWu Biological Technology Co., Ltd. and precisely integrated into the lentiviral vector by homologous recombination, employing the ClonExpress\u0026reg; Ultra One Step Cloning Kit (Vazyme, Nanjing, China, C115). Point mutations generating the \u003cem\u003eHspA1A\u003c/em\u003e\u003csup\u003eW90F\u003c/sup\u003e and \u003cem\u003eHspA1A\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003eCA\u003c/sup\u003e mutants were introduced via PCR-based site-directed mutagenesis and incorporated into the lentiviral vectors following the same recombination protocol. For lentiviral particle production, the doxycycline-inducible constructs were co-transfected into HEK293T cells alongside the packaging plasmids psPAX2 (Addgene, #12260) and the envelope plasmid pMD2.G (Addgene, #12259). Viral particles were harvested, filtered, and used to transduce target cells. Finally, stable cell lines expressing doxycycline-inducible wild-type \u003cem\u003eHspA1A\u003c/em\u003e or its mutants were subsequently established and maintained through rigorous puromycin-based (MedChemExpress, HY-B1743) selection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eReal-time PCR\u003c/h2\u003e \u003cp\u003eTotal mRNA was extracted from mouse tissue samples and cultured cells using the AxyPrep\u0026trade; Multisource Total RNA Miniprep Kit (Axygen, AP-MN-MS-RNA-250), according to the manufacturer's instructions. For tissue samples, specimens underwent multiple freeze-thaw cycles in liquid nitrogen followed by thorough homogenization using a pestle to ensure efficient RNA extraction. The purified RNA samples were reverse-transcribed into complementary DNA (cDNA) utilizing the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen, AT311), which simultaneously eliminates genomic DNA and synthesizes high-quality cDNA. Quantitative PCR (qPCR) assays were conducted with the PerfectStart Green qPCR SuperMix (TransGen, AQ601) following the manufacturer's recommended protocols. Specific primer sequences employed for the qPCR reactions are detailed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Relative mRNA expression levels of target genes were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method with normalization to GAPDH as the housekeeping gene.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHistological Fixation and Staining\u003c/h2\u003e \u003cp\u003eMouse lung tissues were initially fixed in 4% paraformaldehyde (PFA) to preserve tissue morphology. Tissues were then progressively dehydrated through a graded ethanol series (75%, 85%, 95%, and 100%) and cleared in xylene to facilitate paraffin infiltration. Following embedding in paraffin, tissue blocks were sectioned at a uniform thickness of 5 \u0026micro;m using a precision microtome, and the resulting sections were carefully mounted onto clean, adhesive-coated glass slides for further analysis.\u003c/p\u003e \u003cp\u003eFor hematoxylin and eosin (H\u0026amp;E) staining, tissue sections were first deparaffinized in xylene, and then rehydrated through decreasing ethanol concentrations, followed by a brief rinse in distilled water. Sections were subsequently stained sequentially with hematoxylin and then eosin to clearly visualize the general tissue architecture. Finally, the stained sections were dehydrated through an ascending ethanol series, cleared in xylene, and permanently mounted with neutral mounting medium.\u003c/p\u003e \u003cp\u003eMasson\u0026rsquo;s trichrome staining was employed to visualize collagen deposition in lung tissues. Following the same deparaffinization and rehydration procedures described above, tissue sections were stained according to standard Masson\u0026rsquo;s trichrome staining protocols. This method distinctly highlighted collagen fibers, enabling a precise assessment of fibrosis and extracellular matrix remodeling in the tissue samples.\u003c/p\u003e \u003cp\u003e For detection of SA-β-Gal activity, mouse tissues were carefully embedded in optimal cutting temperature (OCT) compound, rapidly snap-frozen in liquid nitrogen to preserve enzyme activity, and subsequently stored at -80\u0026deg;C. Frozen sections (~\u0026thinsp;8 \u0026micro;m thick) were then cut using a cryostat, mounted onto positively charged slides, and stained using the β-galactosidase staining kit (Beyotime Biotechnology, China, C0602) according to the manufacturer's instructions.\u003c/p\u003e \u003cp\u003eAll prepared histological slides were examined and captured using a high-resolution optical microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence Staining\u003c/h2\u003e \u003cp\u003eCultured cells were gently rinsed three times with PBS and fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature. Following fixation, cells were permeabilized with 0.2% Triton X-100 for 10 minutes to facilitate antibody penetration. To minimize nonspecific binding, cells were blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature. Primary antibodies (diluted as specified in Table S2) were then applied, and cells were incubated overnight at 4\u0026deg;C. The next day, cells were thoroughly washed with PBS and incubated with fluorescent-dye conjugated secondary antibodies at room temperature for 1 hour in the dark.\u003c/p\u003e \u003cp\u003eMouse tissue sections were first deparaffinized by immersion in fresh xylene for 10 minutes, repeated twice. Following deparaffinization, the sections were sequentially rehydrated through a graded ethanol series: two washes in 100% ethanol (5 minutes each), followed by successive washes in 95%, 85%, and 75% ethanol (3 minutes each), and finally rinsed thoroughly in distilled water. Antigen retrieval was performed by immersing the slides in Tris-EDTA antigen retrieval buffer (Solarbio, C1038) and heating in a microwave oven at medium power for 15 minutes. After natural cooling to room temperature, the sections were gently washed three times with PBS (5 minutes each). To reduce nonspecific antibody binding, sections were blocked with 5% bovine serum albumin (BSA) in PBS for 1 hour at room temperature. Primary antibodies, (diluted as specified in Table S2) were then applied, and the sections were incubated overnight at 4\u0026deg;C in a humidified chamber. The following day, sections were thoroughly washed three times with PBS (5 minutes per wash) to remove unbound antibodies, and then incubated with fluorescent-dye conjugated secondary antibodies (as detailed in Table S2) at room temperature for 1 hour in the dark. Finally, after additional PBS washes to remove excess secondary antibody, the stained sections were mounted with antifade mounting medium.\u003c/p\u003e \u003cp\u003eHigh-resolution immunofluorescence images were captured using a Nikon A1R HD25 laser scanning confocal microscope, enabling precise visualization and quantification of protein localization and expression patterns in cell and tissue samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eCultured cells and mouse tissue samples were lysed thoroughly using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, China, P0013B) freshly supplemented with Protease and Phosphatase Inhibitor Cocktail (Beyotime Biotechnology, China, P1045) to preserve protein and its phosphorylation. Samples were incubated on ice for 30 minutes with intermittent gentle mixing to ensure complete lysis and extraction. Following incubation, lysates were clarified by centrifugation at 12,000 \u0026times; g for 15 minutes at 4\u0026deg;C. The supernatants containing the extracted proteins were then carefully collected, and protein concentrations were quantified using the Bicinchoninic Acid (BCA) Protein Assay Kit (Beyotime Biotechnology, China, P0399).\u003c/p\u003e \u003cp\u003eFor Western blot analysis, equal amounts of protein (typically 20\u0026ndash;40 \u0026micro;g per lane) were loaded onto SDS-PAGE gels and separated by electrophoresis. Proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes (Merck, ISEQ00010). Membranes were blocked for 1 hour at room temperature using QuickBlock\u0026trade; Western Blocking Buffer (Beyotime Biotechnology, China, P0252) to reduce nonspecific antibody binding. After blocking, membranes were incubated overnight at 4\u0026deg;C with specific primary antibodies diluted in QuickBlock\u0026trade; Western Primary Antibody Dilution Buffer (Beyotime Biotechnology, China, P0256), as detailed in Table S2.\u003c/p\u003e \u003cp\u003eThe next day, membranes were extensively washed with Tris-buffered saline containing 0.1% Tween-20 (TBST) to remove unbound primary antibodies. They were then incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (listed in Table S2) for 1 hour at room temperature. After additional TBST washes, protein bands were visualized using an enhanced chemiluminescence (ECL) detection system (Yeasen Biotechnology, Shanghai, China, 36208ES76). Images were subsequently captured and analyzed using the Amersham Imager 600 imaging system (GE Healthcare) to ensuring high-resolution and accurate protein quantification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003ePulmonary Function Testing\u003c/h2\u003e \u003cp\u003eMice under normal physiological conditions were analyzed using a whole-body plethysmography system (WBP, emka Technologies). Each mouse was individually placed into the plethysmograph chamber for a minimum of 10 minutes to allow for acclimation and stabilization of normal respiratory patterns. Following this habituation period, various respiratory parameters were continuously measured and recorded, including Inspiratory Time (Ti), Expiratory Time (Te), Peak Inspiratory Flow (PIF), Peak Expiratory Flow (PEF), Minute Volume (MV), Breathing Frequency (F), and Relaxation Time (RT). To account for differences in body size and metabolic status among the mice, MV was normaizled to body weight (MV/BW), providing a more accurate and physiologically meaningful assessment of pulmonary ventilation efficiency.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMicroCT Scanning\u003c/h2\u003e \u003cp\u003eLung imaging of mice was performed using a Quantum GX MicroCT imaging system (PerkinElmer, MA, USA) to obtain high-resolution visualization of pulmonary structures. The scanning parameters were carefully optimized to ensure high image quality, including an X-ray voltage of 90 kV, an X-ray current of 88 \u0026micro;A, a field of view (FOV) of 45 mm, and a voxel resolution with a pixel size of 90 \u0026micro;m. Prior to scanning, mice were deeply anesthetized and meticulously positioned on the scanning platform to minimize motion artifacts. Sequential lung image datasets were automatically acquired during the scan, and upon completion, the data were processed for reconstruction and segmentation to generate clear anatomical images of the lung.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative and Statistical analysis\u003c/h2\u003e \u003cp\u003eImageJ software (National Institutes of Health, Bethesda, MD, USA) was utilized to quantify mean fluorescence intensities from immunofluorescence images, and to measure the grayscale values of protein bands from western blot analyses. Graphs illustrating these quantitative results were generated using GraphPad Prism software (GraphPad Software, San Diego, CA, USA). Statistical tests were performed using appropriate tests based on experimental design: Student's t-test was applied for pairwise comparisons between two groups, and one-way analysis of variance (ANOVA) followed by Tukey's post hoc test was conducted for comparisons among three or more groups. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical significance was defined as *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001. All experiments were independently repeated at least three times to ensure robustness, accuracy, and reproducibility of the findings.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Imaging Core Facility, Technology Center for Protein Sciences, Tsinghua University for the assistance in using Nikon A1R HD25 laser confocal microscope, and the Laboratory Animal Research Center, Tsinghua University for the assistance in animal experiments. This work is supported by the National Key R\u0026amp;D Program of China (2022YFA1103704 to S.D.), Beijing Natural Science Foundation (JQ22016 to T.H.M.), the New Cornerstone Investigator Program (to S.D.), and Tsinghua-Peking joint Center for Life Sciences (to S.D.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.L.F., T.H.M and S.D. designed the project, discussed the results, and approved the final version of the manuscript. Y.L.F. performed the experiments and analyzed the data, wrote the manuscript, and supervised the project. F.F.J. and Y.X.Z participated in the animal experiments. Y.L.F., Y.Z., F.F.J., T.H.M and S.D. participated in the discussion of results. S.D. is the lead contact.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared no potential conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of generative AI and AI-assisted technologies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the preparation of this work the authors used ChatGPT in order to improve the readability and quality of the text. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMylonas, A. \u0026amp; O'Loghlen, A. Cellular Senescence and Ageing: Mechanisms and Interventions. \u003cem\u003eFront Aging\u003c/em\u003e 3, 866718 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVarela-Eirin, M. \u0026amp; Demaria, M. 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However, the development of selective senolytic agents that exclusively target SnCs remains a challenge due to the absence of mechanisms that distinctly differentiate them from non-senescent cells (non-SnCs). In this study, we report a serendipitous discovery that Pifithrin-\u0026micro; (PES), through its inhibition of Hsp70, exhibits highly selective senolytic activity. Mechanistic investigations revealed that SnCs harbor dysfunctional lysosomes with compromised membrane integrity\u0026mdash;a vulnerability typically counteracted by the upregulation and lysosomal recruitment of Hsp70, which facilitates lysosomal repair by enhancing ceramide production. Inhibition of Hsp70 by PES selectively disrupts this repair mechanism, inducing catastrophic lysosomal membrane permeability (LMP) and triggering lysosome-dependent cell death (LDCD) specifically in SnCs. Moreover, PES treatment improved aging-related phenotypes and reversed lung fibrosis in mouse models of irradiation-induced accelerated aging and pulmonary fibrosis, respectively. These findings establish Hsp70 as a novel and transformative senolytic target and suggest that targeting the senescence-specific lysosomal repair pathway could provide a safer, more selective, and efficacious alternative to current senolytic strategies, which primarily target non-specific anti-apoptotic pathways.\u003c/p\u003e","manuscriptTitle":"Discovery of Hsp70-Mediated Lysosomal Repair as a Highly Specific Target for Senolysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-29 05:30:21","doi":"10.21203/rs.3.rs-6662488/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-metabolism","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"natmetab","sideBox":"Learn more about [Nature Metabolism](http://www.nature.com/natmetab/)","snPcode":"","submissionUrl":"","title":"Nature Metabolism","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a1fecb53-0c86-4ca6-b72c-ee5a3733497e","owner":[],"postedDate":"May 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":49164757,"name":"Biological sciences/Cell biology/Senescence"},{"id":49164758,"name":"Biological sciences/Cell biology/Mechanisms of disease"},{"id":49164759,"name":"Biological sciences/Cell biology/Cell death"}],"tags":[],"updatedAt":"2026-04-13T09:16:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-29 05:30:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6662488","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6662488","identity":"rs-6662488","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}