ATF6 shapes PERK and IRE1 signaling dynamics during ER stress

ATF6 shapes PERK and IRE1 signaling dynamics during ER stress | 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 ATF6 shapes PERK and IRE1 signaling dynamics during ER stress Susan Logue, Gideon Ong, Masozi Palata, Suraj Shaji, Joel Pearson This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9373070/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract The Unfolded Protein Response (UPR) is a conserved network of signaling pathways controlled by the Endoplasmic Reticulum (ER) anchored stress sensors IRE1, PERK and ATF6. The UPR’s primary function is to help cells manage and resolve ER stress. Compared with the well-characterized IRE1 and PERK pathways, how ATF6 shapes the wider UPR network is still largely unresolved. Using pharmacological inhibition, genetic knockout, and inducible expression models, we show that ATF6 signaling intersects with both the PERK and IRE1 branches of the UPR. During early ER stress, ATF6 promotes PERK expression, with inhibition or loss of ATF6 lowering PERK levels, while selective induction of active ATF6 drives PERK upregulation. As stress shifts from acute to prolonged exposure, ATF6 signaling helps to dampen IRE1 RNase activity. Cells lacking ATF6 or treated with ATF6 inhibitors exhibit prolonged IRE1 RNase activity, while induction of active ATF6 suppresses IRE1 signaling. Our findings identify an unappreciated role for ATF6 as a temporal modulator of UPR signaling, underscoring the importance of communication between ER stress sensors in fine-tuning adaptive responses that dictate cellular outcomes during ER stress. Biological sciences/Cell biology/Cell signalling/Stress signalling Biological sciences/Cell biology/Proteolysis/Protein quality control Biological sciences/Cell biology/Organelles/Endoplasmic reticulum Endoplasmic Reticulum Stress Unfolded Protein Response ATF6 IRE1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The Endoplasmic Reticulum (ER) is recognized as an important site of protein production and packaging within a cell. To efficiently meet demands placed upon it, the ER requires a constant and plentiful supply of energy. Stressors that negatively impact the ER environment (hypoxia, glucose deprivation) or place excessive demands upon the ER (viral infection), result in a breakdown in ER function. Impaired ER function manifests as the accumulation of unfolded proteins within the ER lumen, a condition referred to as ER stress. The Unfolded Protein Response (UPR), a conserved stress response pathway, controlled by three ER anchored receptors, Inositol-Requiring Enzyme 1 Alpha (IRE1α, here after referred to as IRE1), Protein Kinase R (PKR)-like ER Kinase (PERK) and Activating Transcription Factor 6 (ATF6) is triggered in response to ER stress ( 1 – 4 ). The primary objective of the UPR is to re-establish ER homeostasis by enhancing the refolding of recoverable proteins while directing those beyond repair towards degradation. When ER homeostasis cannot be established, UPR signaling transitions from a pro-survival to a pro-death process ensuring removal of the damaged cell ( 5 ). Upon induction of ER stress, IRE1, PERK and ATF6 trigger a series of dynamic and coordinated signaling pathways. IRE1 undergoes oligomerization leading to trans-autophosphorylation and activation of its RNase activity ( 6 ). Active IRE1 splices a 26 nucleotide intron from X-box binding protein 1 ( XBP1) mRNA which, when religated and translated, produces the transcription factor spliced XBP1 (XBP1s) ( 7 – 9 ). XBP1s promotes the expression of ER chaperone proteins and components of the ER-associated degradation (ERAD) machinery helping to support the adaptive pro-survival phase of the UPR ( 10 ). IRE1 RNase activity also mediates Regulated IRE1 Dependent Decay (RIDD)( 11 ) in which IRE1 targets stem loop structures within selected mRNAs leading to their degradation ( 12 ). The role of RIDD within UPR signaling is dynamic; RIDD mediated degradation of mRNAs destined for folding and packaging within the ER helps to avoid further demands being placed on an already compromised ER, while RIDD mediated degradation of Death Receptor 5 ( DR5 ) has been demonstrated to restrain induction of ER mediated cell death signaling ( 13 , 14 ). While IRE1 RNase activity is undoubtedly the most characterized output of IRE1, recent findings have also highlighted emerging roles for IRE1 kinase activity, with IRE1 mediated phosphorylation of the RNA binding protein Pumillo shown to protect XBP1s mRNA from RIDD mediated degradation ( 15 ). Similar to IRE1, induction of ER stress triggers dimerization and trans-autophosphorylation of PERK, which phosphorylates serine 51 on eukaryotic initiation factor 2 alpha (eIF2α), thereby initiating a stall in general cap dependent translation ( 16 , 17 ). While widespread, this translational block is not absolute, as genes such as Activating Transcription Factor 4 (ATF4), that have upstream open reading frames within their 5’ untranslated regions are preferentially translated under these conditions ( 18 ). Once translated, ATF4 increases the expression of genes implicated in processes such as amino acid metabolism and influences cell fate by controlling expression of the pro-apoptotic transcription factor DNA-Damage Inducible Transcript 3 (DDIT3) ( 17 , 19 – 21 ). While the impact of IRE1 and PERK signaling upon ER stress responses has been extensively characterized, the UPR is also modulated by a third less characterized stress sensor, ATF6. ATF6 is a type II transmembrane protein that is constitutively expressed in two forms, ATF6α and ATF6β ( 4 , 22 ). Both ATF6α and β share conserved DNA Binding and b-ZIP domains, but differ in their Transcriptional Activation Domain (TAD), with an eight amino acid sequence essential for maximal transcriptional activity absent within the TAD of ATF6β ( 23 ). Upon induction of ER stress, ATF6α and β are transported from the ER to the Golgi where they undergo processing via site-1 and site-2-proteases resulting in the generation of ATF6 nuclear (ATF6N)( 24 ). While both ATF6α and β are processed, only ATF6αN has transcriptional activity and has been linked to the upregulation of unspliced XBP1 ( XBP1u ) and ER chaperones in particular HSPA5 ( 9 , 25 ). The IRE1 and PERK mediated branches of the UPR have been extensively studied using genetic and pharmacological strategies. However, much less information is available regarding the ATF6 branch. The recent development and characterization of the Ceapin family of inhibitors provides a means to explore ATF6 signaling ( 26 ). In this study, by leveraging both pharmacological and genetic strategies, we mapped the impact of interfering with ATF6 signaling upon the wider UPR network. Results Inhibition of ATF6 with Ceapin-A7 reduces PERK expression while increasing XBP1s levels in cells subjected to ER stress To determine if ATF6 dependent signaling impacts the IRE1 or PERK branches of the UPR, MDA-MB-231 cells were treated with the chemical ER stress inducer thapsigargin (Tg) for various times in the presence or absence of the ATF6α inhibitor Ceapin-A7. Ceapin-A7 functions by trapping full length ATF6 (ATF6FL) in ER resident foci, preventing its translocation to the Golgi apparatus and subsequent processing by site-1 and site-2 proteases ( 27 ). Upon Tg treatment, conversion of ATF6 from its full length 90 kDa form to its processed 50 kDa nuclear form (ATF6N) was evident by 3 h, with levels of ATF6N subsiding as ER stress shifted from an acute (0–6 hr) to a chronic (9–24 hr) setting (Fig. 1 A). Combination with Ceapin-A7, while preventing ATF6FL processing in Tg treated cells, was also associated with a loss of ATF6FL expression especially upon prolonged ER stress (Fig. 1 A). While PERK activation, as indicated by an upshift in PERK protein mobility upon Tg treatment, was not impacted by Ceapin-A7 the level of PERK expression appeared reduced in cells treated with a Tg/Ceapin-A7 combination. This pattern of reduced PERK expression was more pronounced at earlier timepoints (3, 6, 9 h Tg) and dissipated as cells transitioned to a chronic ER stress (Fig. 1 A). IRE1 protein, while increased by Tg treatment, was not altered in Tg/Ceapin-A7 treated cells (Fig. 1 A). However, the expression of the IRE1 effector XBP1s was significantly elevated in cells treated with Tg plus Ceapin-A7 when compared to Tg alone (Fig. 1 A, B). In addition to MDA-MB-231 cells, MCF10a and THP-1 cells displayed similar patterns, with Ceapin-A7 addition enhancing Tg mediated increases in XBP1s, while reducing expression of PERK (Fig. 1 C, D). ATF6 signaling helps to attenuate IRE1 RNase activity To validate observations generated with Ceapin-A7, ATF6α knockout (KO) (here after referred to as ATF6KO) MDA-MB-231 and MCF10a cells were generated via CRISPR/Cas9. Successful editing of the ATF6 locus was verified by Sanger sequencing ( Supplemental Fig. 1 ). As shown in Fig. 2 , while ATF6 expression and processing was readily evident in Tg treated non-targeting (NT) control cells, both ATF6KO MDA-MB-231 and MCF10a cells lacked full length ATF6 and failed to display ATF6N upon Tg treatment (Fig. 2 A, C ) . To validate loss of ATF6 signaling, expression of the ATF6N target GRP78 was assessed by KDEL immunoblotting. Although basal GRP78 expression was not significantly altered in ATF6KO cells, upon induction of ER stress GRP78 expression was blunted in ATF6KO cells when compared to NT controls (Fig. 2 B, D). Similar to Ceapin-A7 treatment, cells lacking ATF6 expression exhibited increased XBP1s upon exposure to ER stress triggered by chemical (Tg) or physiological (serum deprivation) inducers (Fig. 3 A, B, C). To determine whether the increased XBP1s expression observed upon inhibition of ATF6 signaling in Tg treated cells was selective to XBP1s or reflective of a general increase in IRE1 RNase activity, NT and ATF6KO MDA-MB-231 cells were transduced with a baculovirus expressing an IRE1 reporter. Transduced cells constitutively expressed red fluorescent protein, with green fluorescent protein produced only upon activation of IRE1 RNase activity. Using the Incucyte imaging system, the ratio of green to red fluorescence in cells treated with vehicle or Tg was quantitatively monitored, providing a readout of endogenous IRE1 RNase activity. Upon Tg treatment, both NT and ATF6KO MDA-MB-231 cells initially displayed similar IRE1 splicing kinetics, with peak activity occurring in both lines around 12 h (Fig. 4 A). By 24 h, NT cells exhibited a decline in IRE1 RNase activity which was not evident in ATF6KO cells (Fig. 4 A, B). Addition of the IRE1 inhibitor MKC8866 to Tg treated ATF6KO cells suppressed Tg induced IRE1-mediated splicing of the biosensor verifying assay functionality ( Supplemental Fig. 2 ). Collectively, this data indicates that upon exposure to chronic ER stress conditions, loss of ATF6 signaling results in sustained IRE1 RNase activity. As IRE1 RNase activity has two signaling outputs - IRE1-XBP1s and IRE1-RIDD - we next asked if loss of ATF6 signaling impacted both IRE1-XBP1s and IRE1-RIDD equally. To tease apart IRE1–XBP1s and IRE1–RIDD signaling, levels of endogenous XBP1s and the RIDD substrate DGAT2 ( 28 ) were examined. Analysis of IRE1–RIDD activity indicated elevated basal RIDD signaling, as determined by lower DGAT2 expression in ATF6KO cells compared to NT controls (Fig. 4 C). Following induction of ER stress, DGAT2 transcript decreased in both NT and ATF6KO cells (Fig. 4 C). XBP1s expression, while similar at a basal level in both NT and ATF6KO cells, was significantly increased in Tg treated ATF6KO cells when compared to NT controls (Fig. 4 D). Since loss of ATF6 signaling enhances IRE1 RNase activity, we next asked whether selectively overexpressing ATF6N could reduce IRE1 RNase activity. To test this, ATF6N Tet−on MDA-MB-231 cells were generated. Doxycycline (Dox) (1 µg/ml) addition induced expression of ATF6N (Fig. 5 A), which in turn triggered a significant increase in the expression of ATF6N target genes, HSPA5 , XBP1u , and HERPUD1 , confirming the functionality of the inducible system (Fig. 5 B). To assess the impact of ATF6N overexpression upon IRE1 RNase activity, ATF6N Tet−on MDA-MB-231 cells were transduced with the IRE1 reporter construct and the impact of Dox-induced ATF6N expression upon IRE1 RNase signaling assessed. Selective induction of ATF6N reduced IRE1 RNase reporter splicing under both basal conditions and Tg induced ER stress (Fig. 5 C, E, F). Analysis of XBP1s protein expression in Tg treated ATF6N Tet−on MDA-MB-231 cells revealed a similar pattern, with induction of ATF6N reducing Tg-induced XBP1s expression (Fig. 5 D). Collectively, these observations establish a regulatory relationship between ATF6 and IRE1, wherein loss of ATF6 signaling prolongs IRE1 RNase activity, while overexpression of ATF6N suppresses it. ATF6 signaling promotes PERK expression during early ER stress In addition to elevated XBP1s expression, a decrease in PERK protein expression, especially at timepoints associated with early ER stress (3, 6, 9 h Tg), was evident in Tg plus Ceapin-A7 treated cells (Fig. 1 A, C, D). To verify ATF6 mediated regulation of PERK, we assessed PERK expression following ER stress induction in ATF6KO cells. In both ATF6KO MDA-MB-231 (Fig. 6 A) and MCF10a (Fig. 6 B) cells, PERK expression was reduced compared to NT control cells following 6 h Tg treatment, validating earlier results obtained using Ceapin-A7. To further characterize the relationship between ATF6 signaling and PERK, ATF6N Tet−on MDA-MB-231 cells were utilized to examine the outcome of selectively activating ATF6N upon PERK expression. Although ATF6N overexpression suppresses IRE1 RNase activity in MDA-MB-231 cells (Fig. 5 ), given that our previous work has linked XBP1s to transcriptional upregulation of PERK during chronic ER stress ( 29 ), ATF6N mediated changes in PERK transcript were assessed in the presence of the IRE1 inhibitor MKC8866. Both PERK transcript and protein levels significantly increased in ATF6N Tet−on MDA-MB-231 cells treated with Dox (Fig. 6 C, D). The increase in PERK protein expression was accompanied by an upshift in PERK, phosphorylation of eIF2α and elevated ATF4 expression supporting activation of PERK dependent signaling (Fig. 6 D). Given that selective overexpression of ATF6N was able to increase PERK expression, we questioned if PERK is a direct transcriptional target of ATF6N. Chromatin immunoprecipitations (ChIP) using an ATF6 antibody were conducted in MDA-MB-231 cells treated with Tg in the presence and absence of Ceapin-A7. As shown in Fig. 7 , ChIP-qPCR supported increased ATF6 interactions with the PERK promoter of Tg treated cells in a Ceapin-A7 regulated manner, with a similar pattern of regulation observed with the known ATF6N target gene HSPA5 (Fig. 7 A, B). Discussion The UPR comprises of a series of signaling pathways controlled by three ER anchored transmembrane receptors, IRE1, PERK and ATF6. Collectively, IRE1, PERK and ATF6 co-ordinate downstream signaling pathways helping to maintain ER homeostasis. In comparison to the IRE1 and PERK branches, our knowledge of ATF6 and how ATF6 mediated signaling shapes wider UPR networks is limited. In part, this has been due to a lack of reliable ATF6 reagents. However, the recent development of better antibodies, ATF6 inhibitors and ATF6 activators, has sparked renewed interest in this ER stress sensor. In this study, using complementary pharmacological and genetic strategies, we mapped the impact of ATF6 signaling upon the wider UPR network. ATF6 inhibition reduced PERK expression in acute, early ER stress but was associated with sustained IRE1 RNase activity during chronic, long-term ER stress. Given the established role of ATF6 in controlling ER chaperone expression, changes in ATF6 activity could alter global ER stress sensitivity, with loss of ATF6 lowering the threshold for UPR activation, and ATF6 overexpression increasing it. However, our observations argue against this simple threshold model and instead support a more complex regulatory crosstalk between the ATF6 and the IRE1/PERK branches of the UPR. Upon ER stress and UPR induction, PERK transcript and protein is rapidly upregulated ( 29 ). If ATF6 inhibition simply lowers UPR activation thresholds, PERK expression should increase in cells subjected to a combination of ER stress and ATF6 inhibition. Yet we observe the opposite, with loss of ATF6 signaling reducing PERK levels, whereas selective overexpression of ATF6N increases PERK abundance to a level sufficient to trigger downstream signaling. Similar to our observations, selective overexpression of ATF6N was associated with increased PERK signaling in colorectal cancer cells, however, the mechanism underpinning this observation was not determined ( 30 ). Based on our findings, we propose ATF6 controls PERK expression via direct transcriptional regulation. Induction of ATF6N selectively increased PERK mRNA, while chromatin immunoprecipitations confirmed stress-dependent enrichment of ATF6 at the PERK promoter. These findings combined with our previously published work ( 29 ), leads us to propose a model where ATF6N promotes early increases in PERK. As ATF6 signaling dissipates, IRE1-XBP1s mediated regulation helps to maintain PERK expression during long term chronic ER stress underscoring the importance of ER stress sensor communication in shaping UPR network dynamics. While ATF6 inhibition reduced PERK expression during early, acute ER stress, it also associated with a substantial increase in XBP1s levels during prolonged ER stress. A similar elevation in XBP1s was observed in both MDA-MB-231 and MCF10a ATF6KO cells verifying this observation is not selective to Ceapin-A7 but rather an outcome of inhibited ATF6 signaling. Indeed initial, early studies characterizing ATF6 mediated signaling utilizing ATF6α −/− mouse embryonic fibroblasts (MEFs) reported similar observations with sustained splicing of XBP1 observed in ATF6α −/− MEFs subjected to chronic ER stress ( 31 ). Analysis of IRE1 signaling in Tg treated NT versus ATF6KO MDA-MB-231 cells initially demonstrated a similar pattern of IRE1 RNase activity, as determined by normalized green to red intensity. Upon exposure to prolonged ER stress (> 12 h) whereas NT control cells exhibited a reduction in IRE1 signaling, ATF6KO MDA-MB-231 cells displayed a higher, sustained IRE1 activity. If the elevated IRE1 activity observed in ATF6KO cells was solely due to a lower UPR activation threshold, we would expect this difference to be apparent at all time points rather than selectively emerging during chronic ER stress signaling. While loss of ATF6 signaling triggered sustained IRE1 RNase activity during chronic ER stress, overexpression of ATF6N did the opposite and reduced IRE1 RNase activity. Again, this pattern of regulation required a lag phase. In Tg treated cells, ATF6N overexpression did not initially reduce IRE1 activity, with Tg and Tg plus Dox cells displaying similar IRE1 reporter activity, only upon prolonged ER stress (> 12 h) did a reduction in IRE1 activity become apparent. Collectively, our observations suggest that the impact of ATF6 signaling on IRE1 RNase activity is likely more complex than a general increase or decrease in ER capacity and UPR activation thresholds. Similar to our findings, Walter et al observed ATF6 knockdown triggered sustained RNase activity in SH-SY-5Y cells ( 32 ), and more recently work by Tung et al linked increased basal ATF6 signaling in calreticulin deficient CHO-K1 cells to a suppression in XBP1 splicing ( 33 ). Our findings, alongside these prior studies, suggest a model where ATF6N dependent processes trigger a signaling mechanism that may actively restrain IRE1 RNase activity. How IRE1 RNase activity is controlled during ER stress is still somewhat unclear. Several mechanisms have been proposed including RPAP2 mediated dephosphorylation of IRE1 ( 34 ), Sec63 mediated binding of HSPA5 to IRE1 ( 35 ) and direct binding of the protein isomerase PDIA6 to IRE1α ( 36 ). While our findings implicate ATF6 in the regulation of IRE1 RNase activity, defining the precise molecular mechanisms that facilitate this control requires further mechanistic studies. In summary, our work highlights an important role for ATF6 mediated crosstalk in shaping both the PERK and IRE1 signaling branches, providing new insights into ATF6 biology and its broader influence on UPR dynamics. Materials and Methods Antibodies and reagents The following antibodies were used: IRE1 (Cell Signaling Technology, #3294, 1:2000), PERK (Cell Signaling Technology, #3192, 1:5000), ATF6 (Abcam, ab122897, 1:1000), Pan-Actin (Cell Signaling Technology, #8456, 1:5000), Pan-Actin (Cell Signaling Technology, #3700, 1:5000), XBP1s (Cell Signaling Technology #40435, 1:1000), KDEL (Medical ＆ Biological Laboratories Co., Ltd., PM059, 1:5000) , phospho-eIF2α (Cell Signaling Technology, #3398, 1:1000), eIF2α (Cell Signaling Technology, #5324, 1:5000), ATF4 (Cell Signaling Technology, #11815, 1:1000. Thapsigargin (10522) was acquired from Cayman Chemicals. MKC8866 was purchased from AmBeed (A1003533) and Ceapin-A7 from Sigma-Aldrich (SML2330). MG132 was obtained from AdooQ (A11043). All chemicals and inhibitors were resuspended according to manufacturer’s instructions. Cell lines and culturing conditions MDA-MB-231 cells (ATCC, HTB-26) were cultured in high glucose DMEM (Gibco, 11965-092) supplemented with 10% fetal bovine serum (Gibco, 12483-020) and 2 mM GlutaMAX™ (Gibco, 35050-079). MCF10a (A gift from Mowat lab, University of Manitoba) were cultured in HuMEC Basal serum free medium (Gibco, 12753018) to which HuMEC supplement mix (Gibco, 12755013) containing epidermal growth factor, hydrocortisone, isoproterenol, transferrin, and insulin, and 25 mg of bovine pituitary extract was added. THP-1 cells (A gift from the Mookherjee Lab, University of Manitoba) were cultured in RPMI 1640 (Gibco, 11875093) supplemented with 10% fetal bovine serum (Gibco, 12483-020) and 2 mM GlutaMAX™ (Gibco, 35050-079) . All cell lines were cultured at 37°C at 5% CO 2 in a humidified incubator. Cells were routinely split through trypsinization and seeded at an appropriate density 24 hours prior to treatment. Establishment of stable cell lines MDA-MB-231 cells were transduced with lentiviral packaged tetracycline regulatory plasmid, pLV[Exp]-Neo-CMV>Tet3G (Vectorbuilder, USA). Transduced cells were selected by treatment with 900 µg/mL G418 (Sigma Aldrich, G8168). Surviving cells were transduced with lentiviral packaged pLV[Exp]-Puro-TRE3G>V5/{hATF6(1-373aa)} (Vectorbuilder, USA) and selected via culturing in medium supplemented with 1 µg/mL Puromycin (Sigma Aldrich, P8833). To induce ATF6αN expression, stably selected cells were treated with doxycycline (Sigma Aldrich, D9891) at indicated concentrations. CRISPR/Cas9 knockout and validation sgRNA sequences were cloned into the LentiCRISPR v2 vector (Addgene Plasmid #52961) using similar approaches as described (37). Briefly, sgRNA oligonucleotides were annealed and cloned into the BsmBI sites in LentiCRISPR v2 using standard ligation cloning. Lentiviruses were generated using Lenti-X 293T cells and the psPax2 (Addgene plasmid #12260) and pMD2.G (Addgene plasmid #12259) packaging vectors. MDA-MB-231 or MCF10a cells were transduced with virus for 24 h, after which successfully transduced cells were selected via Puromycin (1 µg/mL). Single cell colonies were acquired generated through serial dilution and selection with cloning discs (Sigma, Z374431). To verify knockout of ATF6, genomic DNA was extracted (Monarch Spin gDNA Extraction Kit, T3010S) followed by qPCR amplification with primers flanking the cut-site (Table S1). Synthesized cDNA was purified (Monarch Spin PCR & DNA Cleanup Kit, T1130S) and validated by Sanger sequencing at The Centre for Applied Genomics (Toronto CA). IRE1 RNase Activity Reporter Assay MDA-MB-231 cells were seeded on a 96-well plate and transduced with 5uL (per well) of XBP1-IRE1 Ratiometric Cell Stress Assay Big Sky BacMam vector supplemented with 6mM sodium butyrate (Montana Molecular, U0921G). After 24 hours, cells were subjected to treatment supplemented with 6mM sodium butyrate. IRE1 RNase reporter activity was monitored using the IncuCyte S3 live-cell imaging platform. Images of each well comprising of phase contrast (10x), red channel (500 ms) and green channel (300 ms) were obtained at each scan (1h intervals). Reporter activity (red or green signals) was quantified using the accompanying manufacturer software. IRE1 activity is reported as a ratio of green and red intensity signals. RNA extraction and qPCR Total RNA was isolated using Monarch ® Total RNA Miniprep Kit extraction kit (NEB, T2010S) according to the manufacturer’s protocol. Up to 1 µg of total RNA was reversed transcribed using the SensiFast cDNA synthesis kit (Meridian Bioscience, BIO-65054). qPCR reactions were conducted using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, A25742) and the QuantStudio3 thermocycler system. Annealing/extension reactions were carried out at 60°C for 1 minute followed by denaturation at 95°C for 15 seconds. Primer sequences are listed in Supplementary Table 1 ( Table S1 ). Relative transcript levels were determined using the ΔΔCt method by normalizing target genes against GAPDH (human). The calculated ΔΔCt values were used to assess statistical significance between treatments. Immunoblotting Following treatment, cultured cells were scraped into media on ice. Cells were transferred into a 1.5 mL microcentrifuge tube and washed with ice-cold phosphate-buffered saline (PBS) twice. Whole-cell lysates were prepared using SDS lysis buffer (2% sodium dodecyl sulfate, 50 mM Tris-HCl (pH = 6.8), 0.05% Bromophenol Blue, 10% Glycerol, 5% 2-mercaptoethanol) or radioimmunoprecipitation assay (RIPA) lysis and extraction buffer (Thermo Fisher Scientific, 89900) supplemented with 0.5 mM DTT, 0.1 mM PMSF and HALT™ protease inhibitor cocktail (Thermo Fisher Scientific, 87786). Protein concentration for samples lysed by RIPA buffer were quantified using BCA assay (ThermoFisher, 23225). Lysates were further supplemented with Laemelli buffer (1% SDS, 10% glycerol, 0.02% Bromophenol Blue, 50 mM Tris-HCl (pH = 6.8), 1% 2-mercaptoethanol) and heated at 100°C for 5 minutes. After cooling, protein lysates were loaded onto BioRad Stain-Free™ FastCast™ acrylamide gels (BioRad, #1610183), semi-dry transferred onto 0.2 µm nitrocellulose membranes (BioRad, #1620112) and blocked in PBS-0.1% Tween containing 5% skim milk or EveryBlot blocking buffer (BioRad, #12010020). Chemiluminescent signal was acquired using the ChemiDoc system (BioRad). Chromatin Immunoprecipitation ChIP assays were performed using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, #9003) according to manufacturer’s recommendations. Following treatment, MDA-MB-231 cells were cross-linked with 37% formaldehyde (Sigma Aldrich, F8775) at a final concentration of 1% for 10 min at room temperature. Chromatin was digested by adding 1 µL of micrococcal nuclease (Cell Signaling Technology, #10011) per IP prep and incubation for 20 min at 37 °C. Samples were then subjected to sonication. ChIP was performed using anti-ATF6 [EPR22690-84] - ChIP Grade (Abcam, ab227830, 2µg per 10µg chromatin) or normal rabbit IgG (Cell Signaling Technology, #2729) antibody. Immunoprecipitated DNA fragments were purified and analyzed by qPCR using primers designed against the promoter of PERK or HSPA5 . Results were calculated using the percent input method. The acquired ΔCt values were used to assess statistical significance between treatments. Statistical analysis All data are displayed as mean ± standard deviation (SD) or mean ± standard error of mean (SEM). Statistical analyses were conducted using GraphPad Prism 11. Where appropriate, Student’s t-test or one-way ANOVA followed by Tukey HSD post-Hoc analysis was used to assess statistical significance amongst treatments. Values with P ≤ 0.05 were considered statistically significant. Declarations Data Availability The data supporting the conclusions of this article are included in the article, its supplementary files or available from the corresponding author under reasonable request. Author Contributions GO, MP and SS performed experiments. GO, MP and SEL designed experiments and analyzed data. JP provided expertise and guidance relating to generation and validation of NT and ATF6 knockout cells. GO and SEL prepared the manuscript. SEL devised the study, acquired funding and oversaw the research program. All listed authors reviewed the manuscript and provided critical feedback. Disclosure and competing interests The authors declare no competing interests. Acknowledgements This work was supported by the Canada Research Chairs Program (CRC-2018-00305), NSERC Discovery Grant (RGPIN-2020-04896), Research Manitoba and CFI JELF awards to SEL. GO was supported by a Canada Graduate Doctoral Scholarship (CGSD-588623-2024). We would like to acknowledge the support provided by the QuIPS Platform funded by the CancerCare Manitoba Foundation in aiding the generation of knockout cell lines required for this research. References Cox JS, Shamu CE, Walter P. Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase. Cell. 1993;73(6):1197–206. doi: 10.1016/0092-8674(93)90648-A Mori K, Ma W, Gething MJ, Sambrook J. A transmembrane protein with a cdc2+/CDC28-related kinase activity is required for signaling from the ER to the nucleus. Cell. 1993;74(4):743–56. doi: 10.1016/0092-8674 (93)90521-q PubMed PMID: 8358794. Harding HP, Zhang Y, Ron D. 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Tung J, Huang L, George G, Harding HP, Ron D, Ordóñez A. A genome wide CRISPR/Cas9 screen identifies calreticulin as a selective repressor of ATF6α. eLife. 2024;13. doi: 10.7554/eLife.96979.2 Chang TK, Lawrence DA, Lu M, Tan J, Harnoss JM, Marsters SA, et al. Coordination between Two Branches of the Unfolded Protein Response Determines Apoptotic Cell Fate. Mol Cell. 2018;71(4):629–636.e5. doi: 10.1016/j.molcel.2018.06.038 PubMed PMID: 30118681. Li X, Sun S, Appathurai S, Sundaram A, Plumb R, Mariappan M. A Molecular Mechanism for Turning Off IRE1α Signaling during Endoplasmic Reticulum Stress. Cell Reports. 2020;33(13):108563. doi: 10.1016/j.celrep.2020.108563 Eletto D, Eletto D, Dersh D, Gidalevitz T, Argon Y. Protein Disulfide Isomerase A6 controls the decay of IRE1α signaling via disulfide-dependent association. Mol Cell. 2014;53(4):562–76. doi: 10.1016/j.molcel.2014.01.004 PubMed PMID: 24508390; PubMed Central PMCID: PMC3977204. Sanjana NE, Shalem O, Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods. 2014;11(8):783–4. doi: 10.1038/nmeth.3047 PubMed PMID: 25075903; PubMed Central PMCID: PMC4486245. Additional Declarations (Not answered) Supplementary Files Ongetalsuppltable.docx Supplemental Table 1: List of primer sequences CombineduncroppedblotsCDDSubmission.pdf Uncropped blots figures1.tif Supplemental Data Supplemental Figure 1: Confirmation of single cell clonal ATF6 knockout using Sanger Sequencing. Genomic DNA was extracted from single cell ATF6KO clones of (A) MDA-MB-231 or (B) MCF10a cells. Cut sites were amplified by qPCR producing cDNA which was further purified using a DNA cleanup kit. Sanger sequencing was performed and analyzed using the TIDE: Tracking of Indels by Decomposition online platform. figures2.tif Supplemental Figure 2: Addition of MKC8866 suppresses splicing of the XBP1-IRE1 ratiometric cell stress reporter. Scrambled non-targeting control (NT) MDA-MB-231 cells transduced with a XBP1-IRE1 ratiometric cell stress reporter were treated with combination of Tg (0.5 μM) and MKC8866 (20μM); IRE1 splicing activity quantified (A) over 40 h or (B) following 24 h of treatment. Data shown as mean ± S.E.M. Statistical significance was determined using one-way ANOVA followed by TUKEY HSD post-hoc analysis. p****≤ 0.0001. (C) Representative images of green fluorescent channel after 24 h of treatment. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: revise 27 Apr, 2026 Review # 2 received at journal 26 Apr, 2026 Review # 1 received at journal 23 Apr, 2026 Reviewer # 2 agreed at journal 13 Apr, 2026 Reviewer # 1 agreed at journal 13 Apr, 2026 Reviewers invited by journal 13 Apr, 2026 Submission checks completed at journal 10 Apr, 2026 Editor assigned by journal 09 Apr, 2026 First submitted to journal 09 Apr, 2026 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. We do this by developing innovative software and high quality services for the global research community. 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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-9373070","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":620817972,"identity":"00223fa6-b5ce-4932-b9c4-8448f9300d87","order_by":0,"name":"Susan Logue","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYPACCxDB+ABEsoERYSABIpgNwFrYSNDCBiYZCGmRn5H+8HMBg4ScfETysWreHLs8PvnmYw9+MNjJ49JicCPHWHoGg4Sx4Y20tNu825KL2djY0g17GJING3BpkchhkOZhkEjcOCPHDKiFObGNjcdMgofhACMuLUCHPf4N0ZL/rZh3Wz1Yi+QfhgP2uLQw3EgwA9syXyKHjZl322GwFqDIgUScDjvzxsyax0DC2IDnmbHk3G3HgVrS0o1lDJKTcTqsPf3xbZ4KGzn59uSHH95uq06c33z42MM3FXa2OB0GsQuIDqCLEATy+A0dBaNgFIyCkQwAmfRLFVPqJJoAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-7938-3558","institution":"University of Manitoba","correspondingAuthor":true,"prefix":"","firstName":"Susan","middleName":"","lastName":"Logue","suffix":""},{"id":620817973,"identity":"03d1891a-ca82-4b94-92ff-48c9c0bbc399","order_by":1,"name":"Gideon Ong","email":"","orcid":"","institution":"University of Manitoba","correspondingAuthor":false,"prefix":"","firstName":"Gideon","middleName":"","lastName":"Ong","suffix":""},{"id":620817974,"identity":"001d3e64-0139-467f-8dc1-d549373b99c3","order_by":2,"name":"Masozi Palata","email":"","orcid":"","institution":"University of Manitoba","correspondingAuthor":false,"prefix":"","firstName":"Masozi","middleName":"","lastName":"Palata","suffix":""},{"id":620817975,"identity":"b4d826e3-7594-4c09-8d61-d71c763b751d","order_by":3,"name":"Suraj Shaji","email":"","orcid":"","institution":"University of Manitoba","correspondingAuthor":false,"prefix":"","firstName":"Suraj","middleName":"","lastName":"Shaji","suffix":""},{"id":620817976,"identity":"ca3429ca-d0e6-4254-b343-5e9b7b5d786c","order_by":4,"name":"Joel Pearson","email":"","orcid":"","institution":"University of Manitoba","correspondingAuthor":false,"prefix":"","firstName":"Joel","middleName":"","lastName":"Pearson","suffix":""}],"badges":[],"createdAt":"2026-04-10 00:50:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9373070/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9373070/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107458322,"identity":"8fa7ef1a-bc92-41bf-ba34-f2189e4f6b70","added_by":"auto","created_at":"2026-04-21 16:19:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1347954,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of ATF6 with Ceapin-A7 reduces PERK levels while increasing XBP1s expression in cells subjected to ER stress.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMDA-MB-231 cells were treated with Tg (0.5 μM) alone or in combination with Ceapin-A7 (10 μM) for the indicated timepoints after which cell lysates were collected and immunoblotted for (\u003cstrong\u003eA\u003c/strong\u003e) IRE1, XBP1s, PERK, and ATF6. Actin was used as a loading control. Blots are representative of N=3. Arrow indicates IRE1 specific band. (\u003cstrong\u003eB)\u003c/strong\u003e MDA-MB-231 cells were treated with Tg (0.5 μM) alone or in combination with Ceapin-A7 (10 μM) for 18 h following which RNA was extracted and relative changes in \u003cem\u003eXBP1s\u003c/em\u003e assessed via qPCR. Mean relative expression ± SD, reference gene \u003cem\u003eGAPDH\u003c/em\u003e, N= 3. Statistical significance was determined using one-way ANOVA followed by TUKEY HSD post-hoc analysis. *p ≤ 0.05. MCF10a (\u003cstrong\u003eC\u003c/strong\u003e) or THP-1 (\u003cstrong\u003eD\u003c/strong\u003e) cells were treated with Tg (0.5 μM) alone or in combination with Ceapin-A7 (10 μM) for 6 h with MG132 (10 μM) added during the last hour of treatment to aid detection of ATF6N. Cell lysates were harvested and immunoblotted for XBP1s, PERK, and ATF6. Actin was used as a loading control. Blots are representative of N=3.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9373070/v1/67cc81d8ddea360f3e3200ac.png"},{"id":107490330,"identity":"7f082f9e-8e55-42bd-884f-08e9205b5db6","added_by":"auto","created_at":"2026-04-22 02:51:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":686991,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eATF6a knockout MDA-MB-231 and MCF10a cell validation\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eScrambled non-targeting control (NT) and ATF6aknockout (ATF6KO) MDA-MB-231 and MCF10a cells were treated with Tg (0.5 μM) for 6 h after which cells were harvested and immunoblotted for (\u003cstrong\u003eA, C\u003c/strong\u003e) ATF6 or (\u003cstrong\u003eB, D\u003c/strong\u003e) KDEL (arrow denotes GRP78). Actin was used as a loading control. Blots are representative of N=3.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9373070/v1/3995d51b9378d944d4e16e8d.png"},{"id":107458327,"identity":"93057c7b-0ca4-4268-83fc-e39faef39778","added_by":"auto","created_at":"2026-04-21 16:19:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":664799,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of ATF6 associates with elevated XBP1s expression upon induction of ER stress.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eScrambled non-targeting control (NT) and ATF6aknockout (ATF6KO) (\u003cstrong\u003eA\u003c/strong\u003e) MDA-MB-231 cells and (\u003cstrong\u003eC\u003c/strong\u003e) MCF10a cells were treated with Tg (0.5mM) for 6 or 18 h after which cells were harvested and immunoblotted for XBP1s. (\u003cstrong\u003eB\u003c/strong\u003e) NT and ATF6KO MDA-MB-231 cells were cultured in either full (10%) or reduced serum (2.5%) for 72 h after which cells were harvested and immunoblotted for XBP1s. (\u003cstrong\u003eA-C\u003c/strong\u003e) Actin was used as a loading control. Blots are representative of N=3.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9373070/v1/8f3bf37c197ef2c323de9574.png"},{"id":107458329,"identity":"9d39f359-7c08-4820-a0d1-34b51af3bbc6","added_by":"auto","created_at":"2026-04-21 16:19:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":829098,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of ATF6 associates with prolonged IRE1 RNase activity in cells subjected to ER stress.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNT and ATF6KO MDA-MB-231 cells transduced with a XBP1-IRE1 ratiometric cell stress reporter were treated with Tg (0.5 μM) and IRE1 splicing activity quantified (\u003cstrong\u003eA\u003c/strong\u003e) over 40 h or (\u003cstrong\u003eB\u003c/strong\u003e) following 24 h of treatment. Data shown as mean ± S.E.M. Statistical significance was determined using one-way ANOVA followed by TUKEY HSD post-hoc analysis. *p ≤ 0.05, ***≤ 0.001. (\u003cstrong\u003eC, D\u003c/strong\u003e) NT and ATF6KO MDA-MB-231 cells were treated with Tg (0.5 μM, 18 h) following which RNA was extracted and relative changes in (\u003cstrong\u003eC)\u003c/strong\u003e \u003cem\u003eDGAT2\u003c/em\u003e (N=3) and (\u003cstrong\u003eD)\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eXBP1s\u003c/em\u003e(N=4) assessed. Mean relative expression ± SD, reference gene \u003cem\u003eGAPDH\u003c/em\u003e. Statistical significance was determined using one-way ANOVA followed by TUKEY HSD post-hoc analysis. *p 0.05, *** ≤ 0.001.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-9373070/v1/05ac0d93c835245fcab89166.png"},{"id":107458331,"identity":"b0743ead-3109-41d3-94da-fb68524fb913","added_by":"auto","created_at":"2026-04-21 16:19:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1430583,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of ATF6N suppresses IRE1 RNase activity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMDA-MB-231 ATF6N\u003csup\u003eTet on\u003c/sup\u003e cells were treated with Doxycycline (Dox, 1 μg/mL, 24 h) (\u003cstrong\u003eA\u003c/strong\u003e) alone, (\u003cstrong\u003eB\u003c/strong\u003e) in combination with MKC8866 (20 mM) or (\u003cstrong\u003eD\u003c/strong\u003e) a combination of Doxycycline (Dox, 1 μg/mL) and Tg (0.5 μM) for 6 h. Cells were harvested and (\u003cstrong\u003eA, D\u003c/strong\u003e) immunoblotted ATF6 and XBP1s or (\u003cstrong\u003eB\u003c/strong\u003e) relative changes in \u003cem\u003eHSPA5\u003c/em\u003e, \u003cem\u003eHERPUD1\u003c/em\u003e and \u003cem\u003eXBP1u\u003c/em\u003e (N=3) assessed. (\u003cstrong\u003eA, D\u003c/strong\u003e) Arrow denotes V5-ATF6N, asterisk denotes ATF6N. Actin was used as a loading control. Blots are representative of N=3. (\u003cstrong\u003eB\u003c/strong\u003e) Mean relative expression ± SD, reference gene \u003cem\u003eGAPDH\u003c/em\u003e. Statistical significance was determined using Student’s t-test. *p ≤ 0.05, *** ≤ 0.001. (\u003cstrong\u003eC, E, F\u003c/strong\u003e) MDA-MB-231 ATF6N\u003csup\u003eTet on\u003c/sup\u003e transduced with XBP1-IRE1 ratiometric cell stress reporter were treated with Doxycycline (Dox, 1 μg/mL) (\u003cstrong\u003eC\u003c/strong\u003e) alone or (\u003cstrong\u003eE, F)\u003c/strong\u003e in combination with Tg (0.5 μM) and IRE1 splicing activity quantified (\u003cstrong\u003eC, E\u003c/strong\u003e) over time or (\u003cstrong\u003eF\u003c/strong\u003e) following 24 h treatment. Data shown as mean ± S.E.M. \u003cstrong\u003eS\u003c/strong\u003etatistical significance was determined using one-way ANOVA followed by TUKEY HSD post-hoc analysis. ***p ≤ 0.001, **** ≤ 0.0001.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-9373070/v1/a573bf80735f35e0fedb673c.png"},{"id":107458333,"identity":"a626fb12-50aa-47f3-872b-f9918bd1876e","added_by":"auto","created_at":"2026-04-21 16:19:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":651886,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eATF6 signaling increases PERK expression and downstream signaling.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eScrambled non-targeting control (NT) and ATF6a knockout (ATF6KO) (\u003cstrong\u003eA\u003c/strong\u003e) MDA-MB-231 and (\u003cstrong\u003eB\u003c/strong\u003e) MCF10a cells were treated with Tg (0.5 mM) for 6 h after which cells were harvested and immunoblotted for PERK. MDA-MB-231 ATF6N\u003csup\u003eTet on\u003c/sup\u003e cells were treated with Doxycycline (Dox, 1 μg/mL, 24 h) (\u003cstrong\u003eD\u003c/strong\u003e) alone or (\u003cstrong\u003eC\u003c/strong\u003e) in the presence of MKC8866 (20 mM),\u003cstrong\u003e \u003c/strong\u003efollowing which cells were harvested and (\u003cstrong\u003eC\u003c/strong\u003e) relative changes in \u003cem\u003ePERK\u003c/em\u003e (N=3) assessed or (\u003cstrong\u003eD\u003c/strong\u003e) immunoblotted for PERK, p-eIF2a, eIF2a, ATF4, ATF6. (\u003cstrong\u003eC\u003c/strong\u003e) Mean relative expression ± SD, reference gene \u003cem\u003eGAPDH\u003c/em\u003e. Statistical significance was determined using Student’s t-test. ***p ≤ 0.001. (\u003cstrong\u003eA, B, D)\u003c/strong\u003e Actin was used as a loading control. Blots are representative of N=3.\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-9373070/v1/0ca697029d3e173418c43eef.png"},{"id":107458332,"identity":"2b398031-b3e3-45b6-9356-da05ad0b9174","added_by":"auto","created_at":"2026-04-21 16:19:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":353871,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eER stress induction stimulates ATF6 binding to the promoters of downstream target genes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChromatin immunoprecipitations were performed with cross-linked chromatin from MDA-MB-231 cells treated with Tg (0.5 μM) alone or in combination with Ceapin-A7 (10 μM) for 6 h using either ATF6 or control rabbit IgG antibodies. The enriched DNA was quantified by qPCR using primers designed against (\u003cstrong\u003eA\u003c/strong\u003e) the promoter of \u003cem\u003ePERK\u003c/em\u003e or (\u003cstrong\u003eB\u003c/strong\u003e) promoter of \u003cem\u003eHSPA5\u003c/em\u003e. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin which is equivalent to one. Mean percent signal relative to input ± SD, N=3. Statistical significance for all qPCR experiments was determined using one-way ANOVA followed by TUKEY HSD post-hoc analysis. *p ≤ 0.05, ** ≤ 0.01.\u003c/p\u003e","description":"","filename":"figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-9373070/v1/0b7b7d9193968e9beb5fe4cc.png"},{"id":107490476,"identity":"02a2d398-cc67-462f-aa92-9b6441005964","added_by":"auto","created_at":"2026-04-22 02:52:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6528074,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9373070/v1/457eb816-eb6b-4f15-be05-d567d9d0e59f.pdf"},{"id":107458323,"identity":"48edfea1-7603-468e-9e94-18213898802b","added_by":"auto","created_at":"2026-04-21 16:19:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16248,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Table 1:\u003c/strong\u003e \u003cstrong\u003eList of primer sequences\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Ongetalsuppltable.docx","url":"https://assets-eu.researchsquare.com/files/rs-9373070/v1/4828e40c796e1020c8650b0c.docx"},{"id":107458325,"identity":"4d075b67-734c-4b9d-b040-0ddce186c722","added_by":"auto","created_at":"2026-04-21 16:19:03","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":7239134,"visible":true,"origin":"","legend":"Uncropped blots","description":"","filename":"CombineduncroppedblotsCDDSubmission.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9373070/v1/1060451f8a939e0c256cd44e.pdf"},{"id":107490332,"identity":"12f5870a-1c21-4cae-983c-d72dea681ca0","added_by":"auto","created_at":"2026-04-22 02:51:42","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":15831892,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Figure 1:\u003c/strong\u003e \u003cstrong\u003eConfirmation of single cell clonal ATF6 knockout using Sanger Sequencing\u003c/strong\u003e. Genomic DNA was extracted from single cell ATF6KO clones of (\u003cstrong\u003eA\u003c/strong\u003e) MDA-MB-231 or (\u003cstrong\u003eB\u003c/strong\u003e) MCF10a cells. Cut sites were amplified by qPCR producing cDNA which was further purified using a DNA cleanup kit. Sanger sequencing was performed and analyzed using the TIDE: Tracking of Indels by Decomposition online platform.\u003c/p\u003e","description":"","filename":"figures1.tif","url":"https://assets-eu.researchsquare.com/files/rs-9373070/v1/ee0b1be64428b68018ff18bb.tif"},{"id":107458330,"identity":"42a6ced4-8233-4950-959f-54a0521933c1","added_by":"auto","created_at":"2026-04-21 16:19:04","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":19335152,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Figure 2:\u003c/strong\u003e \u003cstrong\u003eAddition of MKC8866 suppresses splicing of the XBP1-IRE1 ratiometric cell stress reporter\u003c/strong\u003e. Scrambled non-targeting control (NT) MDA-MB-231 cells transduced with a XBP1-IRE1 ratiometric cell stress reporter were treated with combination of Tg (0.5 μM) and MKC8866 (20μM); IRE1 splicing activity quantified (\u003cstrong\u003eA\u003c/strong\u003e) over 40 h or (\u003cstrong\u003eB\u003c/strong\u003e) following 24 h of treatment. Data shown as mean ± S.E.M. \u003cstrong\u003eS\u003c/strong\u003etatistical significance was determined using one-way ANOVA followed by TUKEY HSD post-hoc analysis. p****≤ 0.0001. \u003cstrong\u003e(C) \u003c/strong\u003eRepresentative images of green fluorescent channel after 24 h of treatment.\u003c/p\u003e","description":"","filename":"figures2.tif","url":"https://assets-eu.researchsquare.com/files/rs-9373070/v1/17955065cc1a0d1c4aae70fa.tif"}],"financialInterests":"(Not answered)","formattedTitle":"ATF6 shapes PERK and IRE1 signaling dynamics during ER stress","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe Endoplasmic Reticulum (ER) is recognized as an important site of protein production and packaging within a cell. To efficiently meet demands placed upon it, the ER requires a constant and plentiful supply of energy. Stressors that negatively impact the ER environment (hypoxia, glucose deprivation) or place excessive demands upon the ER (viral infection), result in a breakdown in ER function. Impaired ER function manifests as the accumulation of unfolded proteins within the ER lumen, a condition referred to as ER stress. The Unfolded Protein Response (UPR), a conserved stress response pathway, controlled by three ER anchored receptors, Inositol-Requiring Enzyme 1 Alpha (IRE1α, here after referred to as IRE1), Protein Kinase R (PKR)-like ER Kinase (PERK) and Activating Transcription Factor 6 (ATF6) is triggered in response to ER stress (\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). The primary objective of the UPR is to re-establish ER homeostasis by enhancing the refolding of recoverable proteins while directing those beyond repair towards degradation. When ER homeostasis cannot be established, UPR signaling transitions from a pro-survival to a pro-death process ensuring removal of the damaged cell (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUpon induction of ER stress, IRE1, PERK and ATF6 trigger a series of dynamic and coordinated signaling pathways. IRE1 undergoes oligomerization leading to trans-autophosphorylation and activation of its RNase activity (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Active IRE1 splices a 26 nucleotide intron from \u003cem\u003eX-box binding protein 1\u003c/em\u003e (\u003cem\u003eXBP1)\u003c/em\u003e mRNA which, when religated and translated, produces the transcription factor spliced XBP1 (XBP1s) (\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). XBP1s promotes the expression of ER chaperone proteins and components of the ER-associated degradation (ERAD) machinery helping to support the adaptive pro-survival phase of the UPR (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). IRE1 RNase activity also mediates Regulated IRE1 Dependent Decay (RIDD)(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) in which IRE1 targets stem loop structures within selected mRNAs leading to their degradation (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). The role of RIDD within UPR signaling is dynamic; RIDD mediated degradation of mRNAs destined for folding and packaging within the ER helps to avoid further demands being placed on an already compromised ER, while RIDD mediated degradation of \u003cem\u003eDeath Receptor 5\u003c/em\u003e (\u003cem\u003eDR5\u003c/em\u003e) has been demonstrated to restrain induction of ER mediated cell death signaling (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). While IRE1 RNase activity is undoubtedly the most characterized output of IRE1, recent findings have also highlighted emerging roles for IRE1 kinase activity, with IRE1 mediated phosphorylation of the RNA binding protein Pumillo shown to protect \u003cem\u003eXBP1s\u003c/em\u003e mRNA from RIDD mediated degradation (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSimilar to IRE1, induction of ER stress triggers dimerization and trans-autophosphorylation of PERK, which phosphorylates serine 51 on eukaryotic initiation factor 2 alpha (eIF2α), thereby initiating a stall in general cap dependent translation (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). While widespread, this translational block is not absolute, as genes such as Activating Transcription Factor 4 (ATF4), that have upstream open reading frames within their 5\u0026rsquo; untranslated regions are preferentially translated under these conditions (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Once translated, ATF4 increases the expression of genes implicated in processes such as amino acid metabolism and influences cell fate by controlling expression of the pro-apoptotic transcription factor DNA-Damage Inducible Transcript 3 (DDIT3) (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). While the impact of IRE1 and PERK signaling upon ER stress responses has been extensively characterized, the UPR is also modulated by a third less characterized stress sensor, ATF6.\u003c/p\u003e \u003cp\u003eATF6 is a type II transmembrane protein that is constitutively expressed in two forms, ATF6α and ATF6β (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Both ATF6α and β share conserved DNA Binding and b-ZIP domains, but differ in their Transcriptional Activation Domain (TAD), with an eight amino acid sequence essential for maximal transcriptional activity absent within the TAD of ATF6β (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Upon induction of ER stress, ATF6α and β are transported from the ER to the Golgi where they undergo processing via site-1 and site-2-proteases resulting in the generation of ATF6 nuclear (ATF6N)(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). While both ATF6α and β are processed, only ATF6αN has transcriptional activity and has been linked to the upregulation of unspliced \u003cem\u003eXBP1\u003c/em\u003e (\u003cem\u003eXBP1u\u003c/em\u003e) and ER chaperones in particular \u003cem\u003eHSPA5\u003c/em\u003e (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe IRE1 and PERK mediated branches of the UPR have been extensively studied using genetic and pharmacological strategies. However, much less information is available regarding the ATF6 branch. The recent development and characterization of the Ceapin family of inhibitors provides a means to explore ATF6 signaling (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). In this study, by leveraging both pharmacological and genetic strategies, we mapped the impact of interfering with ATF6 signaling upon the wider UPR network.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eInhibition of ATF6 with Ceapin-A7 reduces PERK expression while increasing XBP1s levels in cells subjected to ER stress\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo determine if ATF6 dependent signaling impacts the IRE1 or PERK branches of the UPR, MDA-MB-231 cells were treated with the chemical ER stress inducer thapsigargin (Tg) for various times in the presence or absence of the ATF6α inhibitor Ceapin-A7. Ceapin-A7 functions by trapping full length ATF6 (ATF6FL) in ER resident foci, preventing its translocation to the Golgi apparatus and subsequent processing by site-1 and site-2 proteases (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Upon Tg treatment, conversion of ATF6 from its full length 90 kDa form to its processed 50 kDa nuclear form (ATF6N) was evident by 3 h, with levels of ATF6N subsiding as ER stress shifted from an acute (0\u0026ndash;6 hr) to a chronic (9\u0026ndash;24 hr) setting (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Combination with Ceapin-A7, while preventing ATF6FL processing in Tg treated cells, was also associated with a loss of ATF6FL expression especially upon prolonged ER stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). While PERK activation, as indicated by an upshift in PERK protein mobility upon Tg treatment, was not impacted by Ceapin-A7 the level of PERK expression appeared reduced in cells treated with a Tg/Ceapin-A7 combination. This pattern of reduced PERK expression was more pronounced at earlier timepoints (3, 6, 9 h Tg) and dissipated as cells transitioned to a chronic ER stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). IRE1 protein, while increased by Tg treatment, was not altered in Tg/Ceapin-A7 treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). However, the expression of the IRE1 effector XBP1s was significantly elevated in cells treated with Tg plus Ceapin-A7 when compared to Tg alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). In addition to MDA-MB-231 cells, MCF10a and THP-1 cells displayed similar patterns, with Ceapin-A7 addition enhancing Tg mediated increases in XBP1s, while reducing expression of PERK (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eATF6 signaling helps to attenuate IRE1 RNase activity\u003c/h2\u003e \u003cp\u003eTo validate observations generated with Ceapin-A7, ATF6α knockout (KO) (here after referred to as ATF6KO) MDA-MB-231 and MCF10a cells were generated via CRISPR/Cas9. Successful editing of the ATF6 locus was verified by Sanger sequencing (\u003cb\u003eSupplemental Fig.\u0026nbsp;1\u003c/b\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, while ATF6 expression and processing was readily evident in Tg treated non-targeting (NT) control cells, both ATF6KO MDA-MB-231 and MCF10a cells lacked full length ATF6 and failed to display ATF6N upon Tg treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, C\u003cb\u003e)\u003c/b\u003e. To validate loss of ATF6 signaling, expression of the ATF6N target GRP78 was assessed by KDEL immunoblotting. Although basal GRP78 expression was not significantly altered in ATF6KO cells, upon induction of ER stress GRP78 expression was blunted in ATF6KO cells when compared to NT controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, D). Similar to Ceapin-A7 treatment, cells lacking ATF6 expression exhibited increased XBP1s upon exposure to ER stress triggered by chemical (Tg) or physiological (serum deprivation) inducers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B, C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine whether the increased XBP1s expression observed upon inhibition of ATF6 signaling in Tg treated cells was selective to XBP1s or reflective of a general increase in IRE1 RNase activity, NT and ATF6KO MDA-MB-231 cells were transduced with a baculovirus expressing an IRE1 reporter. Transduced cells constitutively expressed red fluorescent protein, with green fluorescent protein produced only upon activation of IRE1 RNase activity. Using the Incucyte imaging system, the ratio of green to red fluorescence in cells treated with vehicle or Tg was quantitatively monitored, providing a readout of endogenous IRE1 RNase activity. Upon Tg treatment, both NT and ATF6KO MDA-MB-231 cells initially displayed similar IRE1 splicing kinetics, with peak activity occurring in both lines around 12 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). By 24 h, NT cells exhibited a decline in IRE1 RNase activity which was not evident in ATF6KO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). Addition of the IRE1 inhibitor MKC8866 to Tg treated ATF6KO cells suppressed Tg induced IRE1-mediated splicing of the biosensor verifying assay functionality (\u003cb\u003eSupplemental Fig.\u0026nbsp;2\u003c/b\u003e). Collectively, this data indicates that upon exposure to chronic ER stress conditions, loss of ATF6 signaling results in sustained IRE1 RNase activity. As IRE1 RNase activity has two signaling outputs - IRE1-XBP1s and IRE1-RIDD - we next asked if loss of ATF6 signaling impacted both IRE1-XBP1s and IRE1-RIDD equally. To tease apart IRE1\u0026ndash;XBP1s and IRE1\u0026ndash;RIDD signaling, levels of endogenous \u003cem\u003eXBP1s\u003c/em\u003e and the RIDD substrate \u003cem\u003eDGAT2\u003c/em\u003e (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) were examined. Analysis of IRE1\u0026ndash;RIDD activity indicated elevated basal RIDD signaling, as determined by lower \u003cem\u003eDGAT2\u003c/em\u003e expression in ATF6KO cells compared to NT controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Following induction of ER stress, \u003cem\u003eDGAT2\u003c/em\u003e transcript decreased in both NT and ATF6KO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). \u003cem\u003eXBP1s\u003c/em\u003e expression, while similar at a basal level in both NT and ATF6KO cells, was significantly increased in Tg treated ATF6KO cells when compared to NT controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince loss of ATF6 signaling enhances IRE1 RNase activity, we next asked whether selectively overexpressing ATF6N could reduce IRE1 RNase activity. To test this, ATF6N\u003csup\u003eTet\u0026minus;on\u003c/sup\u003e MDA-MB-231 cells were generated. Doxycycline (Dox) (1 \u0026micro;g/ml) addition induced expression of ATF6N (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), which in turn triggered a significant increase in the expression of ATF6N target genes, \u003cem\u003eHSPA5\u003c/em\u003e, \u003cem\u003eXBP1u\u003c/em\u003e, and \u003cem\u003eHERPUD1\u003c/em\u003e, confirming the functionality of the inducible system (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). To assess the impact of ATF6N overexpression upon IRE1 RNase activity, ATF6N\u003csup\u003eTet\u0026minus;on\u003c/sup\u003e MDA-MB-231 cells were transduced with the IRE1 reporter construct and the impact of Dox-induced ATF6N expression upon IRE1 RNase signaling assessed. Selective induction of ATF6N reduced IRE1 RNase reporter splicing under both basal conditions and Tg induced ER stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, E, F). Analysis of XBP1s protein expression in Tg treated ATF6N\u003csup\u003eTet\u0026minus;on\u003c/sup\u003e MDA-MB-231 cells revealed a similar pattern, with induction of ATF6N reducing Tg-induced XBP1s expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Collectively, these observations establish a regulatory relationship between ATF6 and IRE1, wherein loss of ATF6 signaling prolongs IRE1 RNase activity, while overexpression of ATF6N suppresses it.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eATF6 signaling promotes PERK expression during early ER stress\u003c/h3\u003e\n\u003cp\u003eIn addition to elevated XBP1s expression, a decrease in PERK protein expression, especially at timepoints associated with early ER stress (3, 6, 9 h Tg), was evident in Tg plus Ceapin-A7 treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, C, D). To verify ATF6 mediated regulation of PERK, we assessed PERK expression following ER stress induction in ATF6KO cells. In both ATF6KO MDA-MB-231 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) and MCF10a (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) cells, PERK expression was reduced compared to NT control cells following 6 h Tg treatment, validating earlier results obtained using Ceapin-A7.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further characterize the relationship between ATF6 signaling and PERK, ATF6N\u003csup\u003eTet\u0026minus;on\u003c/sup\u003e MDA-MB-231 cells were utilized to examine the outcome of selectively activating ATF6N upon PERK expression. Although ATF6N overexpression suppresses IRE1 RNase activity in MDA-MB-231 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), given that our previous work has linked XBP1s to transcriptional upregulation of PERK during chronic ER stress (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), ATF6N mediated changes in PERK transcript were assessed in the presence of the IRE1 inhibitor MKC8866. Both PERK transcript and protein levels significantly increased in ATF6N\u003csup\u003eTet\u0026minus;on\u003c/sup\u003e MDA-MB-231 cells treated with Dox (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D). The increase in PERK protein expression was accompanied by an upshift in PERK, phosphorylation of eIF2α and elevated ATF4 expression supporting activation of PERK dependent signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Given that selective overexpression of ATF6N was able to increase PERK expression, we questioned if PERK is a direct transcriptional target of ATF6N. Chromatin immunoprecipitations (ChIP) using an ATF6 antibody were conducted in MDA-MB-231 cells treated with Tg in the presence and absence of Ceapin-A7. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, ChIP-qPCR supported increased ATF6 interactions with the \u003cem\u003ePERK\u003c/em\u003e promoter of Tg treated cells in a Ceapin-A7 regulated manner, with a similar pattern of regulation observed with the known ATF6N target gene \u003cem\u003eHSPA5\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe UPR comprises of a series of signaling pathways controlled by three ER anchored transmembrane receptors, IRE1, PERK and ATF6. Collectively, IRE1, PERK and ATF6 co-ordinate downstream signaling pathways helping to maintain ER homeostasis. In comparison to the IRE1 and PERK branches, our knowledge of ATF6 and how ATF6 mediated signaling shapes wider UPR networks is limited. In part, this has been due to a lack of reliable ATF6 reagents. However, the recent development of better antibodies, ATF6 inhibitors and ATF6 activators, has sparked renewed interest in this ER stress sensor. In this study, using complementary pharmacological and genetic strategies, we mapped the impact of ATF6 signaling upon the wider UPR network. ATF6 inhibition reduced PERK expression in acute, early ER stress but was associated with sustained IRE1 RNase activity during chronic, long-term ER stress. Given the established role of ATF6 in controlling ER chaperone expression, changes in ATF6 activity could alter global ER stress sensitivity, with loss of ATF6 lowering the threshold for UPR activation, and ATF6 overexpression increasing it. However, our observations argue against this simple threshold model and instead support a more complex regulatory crosstalk between the ATF6 and the IRE1/PERK branches of the UPR.\u003c/p\u003e \u003cp\u003eUpon ER stress and UPR induction, PERK transcript and protein is rapidly upregulated (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). If ATF6 inhibition simply lowers UPR activation thresholds, PERK expression should increase in cells subjected to a combination of ER stress and ATF6 inhibition. Yet we observe the opposite, with loss of ATF6 signaling reducing PERK levels, whereas selective overexpression of ATF6N increases PERK abundance to a level sufficient to trigger downstream signaling. Similar to our observations, selective overexpression of ATF6N was associated with increased PERK signaling in colorectal cancer cells, however, the mechanism underpinning this observation was not determined (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Based on our findings, we propose ATF6 controls PERK expression via direct transcriptional regulation. Induction of ATF6N selectively increased PERK mRNA, while chromatin immunoprecipitations confirmed stress-dependent enrichment of ATF6 at the PERK promoter. These findings combined with our previously published work (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), leads us to propose a model where ATF6N promotes early increases in PERK. As ATF6 signaling dissipates, IRE1-XBP1s mediated regulation helps to maintain PERK expression during long term chronic ER stress underscoring the importance of ER stress sensor communication in shaping UPR network dynamics.\u003c/p\u003e \u003cp\u003eWhile ATF6 inhibition reduced PERK expression during early, acute ER stress, it also associated with a substantial increase in XBP1s levels during prolonged ER stress. A similar elevation in XBP1s was observed in both MDA-MB-231 and MCF10a ATF6KO cells verifying this observation is not selective to Ceapin-A7 but rather an outcome of inhibited ATF6 signaling. Indeed initial, early studies characterizing ATF6 mediated signaling utilizing ATF6α\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mouse embryonic fibroblasts (MEFs) reported similar observations with sustained splicing of XBP1 observed in ATF6α\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e MEFs subjected to chronic ER stress (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Analysis of IRE1 signaling in Tg treated NT versus ATF6KO MDA-MB-231 cells initially demonstrated a similar pattern of IRE1 RNase activity, as determined by normalized green to red intensity. Upon exposure to prolonged ER stress (\u0026gt;\u0026thinsp;12 h) whereas NT control cells exhibited a reduction in IRE1 signaling, ATF6KO MDA-MB-231 cells displayed a higher, sustained IRE1 activity. If the elevated IRE1 activity observed in ATF6KO cells was solely due to a lower UPR activation threshold, we would expect this difference to be apparent at all time points rather than selectively emerging during chronic ER stress signaling.\u003c/p\u003e \u003cp\u003eWhile loss of ATF6 signaling triggered sustained IRE1 RNase activity during chronic ER stress, overexpression of ATF6N did the opposite and reduced IRE1 RNase activity. Again, this pattern of regulation required a lag phase. In Tg treated cells, ATF6N overexpression did not initially reduce IRE1 activity, with Tg and Tg plus Dox cells displaying similar IRE1 reporter activity, only upon prolonged ER stress (\u0026gt;\u0026thinsp;12 h) did a reduction in IRE1 activity become apparent. Collectively, our observations suggest that the impact of ATF6 signaling on IRE1 RNase activity is likely more complex than a general increase or decrease in ER capacity and UPR activation thresholds. Similar to our findings, Walter \u003cem\u003eet al\u003c/em\u003e observed ATF6 knockdown triggered sustained RNase activity in SH-SY-5Y cells (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e), and more recently work by Tung \u003cem\u003eet al\u003c/em\u003e linked increased basal ATF6 signaling in calreticulin deficient CHO-K1 cells to a suppression in \u003cem\u003eXBP1\u003c/em\u003e splicing (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Our findings, alongside these prior studies, suggest a model where ATF6N dependent processes trigger a signaling mechanism that may actively restrain IRE1 RNase activity.\u003c/p\u003e \u003cp\u003eHow IRE1 RNase activity is controlled during ER stress is still somewhat unclear. Several mechanisms have been proposed including RPAP2 mediated dephosphorylation of IRE1 (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), Sec63 mediated binding of HSPA5 to IRE1 (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e) and direct binding of the protein isomerase PDIA6 to IRE1α (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). While our findings implicate ATF6 in the regulation of IRE1 RNase activity, defining the precise molecular mechanisms that facilitate this control requires further mechanistic studies. In summary, our work highlights an important role for ATF6 mediated crosstalk in shaping both the PERK and IRE1 signaling branches, providing new insights into ATF6 biology and its broader influence on UPR dynamics.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eAntibodies and reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe following antibodies were used: IRE1 (Cell Signaling Technology, #3294, 1:2000), PERK (Cell Signaling Technology, #3192, 1:5000), ATF6 (Abcam, ab122897, 1:1000), Pan-Actin (Cell Signaling Technology, #8456, 1:5000), Pan-Actin (Cell Signaling Technology, #3700, 1:5000), XBP1s (Cell Signaling Technology #40435, 1:1000), KDEL (Medical\u0026nbsp;＆\u0026nbsp;Biological Laboratories Co., Ltd., PM059, 1:5000) , phospho-eIF2\u0026alpha; (Cell Signaling Technology, #3398, 1:1000), eIF2\u0026alpha; (Cell Signaling Technology, #5324, 1:5000), ATF4 (Cell Signaling Technology, #11815, 1:1000. Thapsigargin (10522) was acquired from Cayman Chemicals. MKC8866 was purchased from AmBeed (A1003533) and Ceapin-A7 from Sigma-Aldrich (SML2330). MG132 was obtained from AdooQ (A11043). All chemicals and inhibitors were resuspended according to manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell lines and culturing conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMDA-MB-231 cells (ATCC, HTB-26) were cultured in high glucose DMEM (Gibco, 11965-092) supplemented with 10% fetal bovine serum (Gibco, 12483-020) and 2 mM GlutaMAX\u0026trade; (Gibco, 35050-079). MCF10a (A gift from Mowat lab, University of Manitoba)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ewere cultured in HuMEC Basal serum free medium (Gibco, 12753018) to which HuMEC supplement mix (Gibco, 12755013) containing epidermal growth factor, hydrocortisone, isoproterenol, transferrin, and insulin, and 25 mg of bovine pituitary extract was added. THP-1 cells (A gift from the Mookherjee\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eLab, University of Manitoba)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ewere cultured in RPMI 1640 (Gibco, 11875093) supplemented with 10% fetal bovine serum (Gibco, 12483-020) and 2 mM GlutaMAX\u0026trade; (Gibco, 35050-079)\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eAll cell lines were cultured at 37\u0026deg;C at 5% CO\u003csub\u003e2\u003c/sub\u003e in a humidified incubator. Cells were routinely split through trypsinization and seeded at an appropriate density 24 hours prior to treatment. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstablishment of stable cell lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMDA-MB-231 cells were transduced with lentiviral packaged tetracycline regulatory plasmid, pLV[Exp]-Neo-CMV\u0026gt;Tet3G (Vectorbuilder, USA). Transduced cells were selected by treatment with 900 \u0026micro;g/mL G418 (Sigma Aldrich, G8168). Surviving cells were transduced with lentiviral packaged pLV[Exp]-Puro-TRE3G\u0026gt;V5/{hATF6(1-373aa)} (Vectorbuilder, USA) and selected via culturing in medium supplemented with 1 \u0026micro;g/mL Puromycin (Sigma Aldrich, P8833). To induce ATF6\u0026alpha;N expression, stably selected cells were treated with doxycycline (Sigma Aldrich, D9891) at indicated concentrations.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRISPR/Cas9 knockout and validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003esgRNA sequences were cloned into the LentiCRISPR v2 vector \u0026nbsp;(Addgene Plasmid #52961) using similar approaches as described\u0026nbsp;(37). Briefly, sgRNA oligonucleotides were annealed and cloned into the BsmBI sites in LentiCRISPR v2 using standard ligation cloning. Lentiviruses were generated using Lenti-X 293T cells and the psPax2 (Addgene plasmid #12260) and pMD2.G (Addgene plasmid #12259) packaging vectors. MDA-MB-231 or MCF10a cells were transduced with virus for 24 h, after which successfully transduced cells were selected via Puromycin (1 \u0026micro;g/mL). Single cell colonies were acquired generated through serial dilution and selection with cloning discs (Sigma, Z374431). To verify knockout of ATF6, genomic DNA was extracted (Monarch Spin gDNA Extraction Kit, T3010S) followed by qPCR amplification with primers flanking the cut-site (Table S1). Synthesized cDNA was purified (Monarch Spin PCR \u0026amp; DNA Cleanup Kit, T1130S) and validated by Sanger sequencing at The Centre for Applied Genomics (Toronto CA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;IRE1 RNase Activity Reporter Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMDA-MB-231 cells were seeded on a 96-well plate and transduced with 5uL (per well) of XBP1-IRE1 Ratiometric Cell Stress Assay Big Sky BacMam vector supplemented with 6mM sodium butyrate (Montana Molecular, U0921G). After 24 hours, cells were subjected to treatment supplemented with 6mM sodium butyrate. IRE1 RNase reporter activity was monitored using the IncuCyte S3 live-cell imaging platform. Images of each well comprising of phase contrast (10x), red channel (500 ms) and green channel (300 ms) were obtained at each scan (1h intervals). Reporter activity (red or green signals) was quantified using the accompanying manufacturer software. IRE1 activity is reported as a ratio of green and red intensity signals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;RNA extraction and qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated using Monarch\u003csup\u003e\u0026reg;\u003c/sup\u003e Total RNA Miniprep Kit extraction kit (NEB, T2010S) according to the manufacturer\u0026rsquo;s protocol. Up to 1 \u0026micro;g of total RNA was reversed transcribed using the SensiFast cDNA synthesis kit (Meridian Bioscience, BIO-65054). qPCR reactions were conducted using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, A25742) and the QuantStudio3 thermocycler system. Annealing/extension reactions were carried out at 60\u0026deg;C for 1 minute followed by denaturation at 95\u0026deg;C for 15 seconds. Primer sequences are listed in Supplementary Table 1 (\u003cstrong\u003eTable S1\u003c/strong\u003e). Relative transcript levels were determined using the \u0026Delta;\u0026Delta;Ct method by normalizing target genes against \u003cem\u003eGAPDH\u003c/em\u003e (human). The calculated \u0026Delta;\u0026Delta;Ct values were used to assess statistical significance between treatments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoblotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing treatment, cultured cells were scraped into media on ice. Cells were transferred into a 1.5 mL microcentrifuge tube and washed with ice-cold phosphate-buffered saline (PBS) twice. \u0026nbsp; Whole-cell lysates were prepared using SDS lysis buffer (2% sodium dodecyl sulfate, 50 mM Tris-HCl (pH = 6.8), 0.05% Bromophenol Blue, 10% Glycerol, 5% 2-mercaptoethanol) or radioimmunoprecipitation assay (RIPA) lysis and extraction buffer (Thermo Fisher Scientific, 89900) supplemented with 0.5 mM DTT, 0.1 mM PMSF and HALT\u0026trade; protease inhibitor cocktail (Thermo Fisher Scientific, 87786). Protein concentration for samples lysed by RIPA buffer were quantified using BCA assay (ThermoFisher, 23225). Lysates were further supplemented with Laemelli buffer (1% SDS, 10% glycerol, 0.02% Bromophenol Blue, 50 mM Tris-HCl (pH = 6.8), 1% 2-mercaptoethanol) and heated at 100\u0026deg;C for 5 minutes. After cooling, protein lysates were loaded onto BioRad Stain-Free\u0026trade; FastCast\u0026trade; acrylamide gels (BioRad, #1610183), semi-dry transferred onto 0.2 \u0026micro;m nitrocellulose membranes (BioRad, #1620112) and blocked in PBS-0.1% Tween containing 5% skim milk or EveryBlot blocking buffer (BioRad, #12010020). Chemiluminescent signal was acquired using the ChemiDoc system (BioRad).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromatin Immunoprecipitation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChIP assays were performed using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, #9003) according to manufacturer\u0026rsquo;s recommendations. Following treatment, MDA-MB-231 cells were cross-linked with 37% formaldehyde (Sigma Aldrich, F8775) at a final concentration of 1% for 10\u0026thinsp;min at room temperature. Chromatin was digested by adding 1\u0026thinsp;\u0026micro;L of micrococcal nuclease (Cell Signaling Technology, #10011) per IP prep and incubation for 20\u0026thinsp;min at 37\u0026thinsp;\u0026deg;C. Samples were then subjected to sonication. ChIP was performed using anti-ATF6 [EPR22690-84] - ChIP Grade (Abcam, ab227830, 2\u0026micro;g per 10\u0026micro;g chromatin) or normal rabbit IgG (Cell Signaling Technology, #2729) antibody. Immunoprecipitated DNA fragments were purified and analyzed by qPCR using primers designed against the promoter of \u003cem\u003ePERK\u003c/em\u003e or \u003cem\u003eHSPA5\u003c/em\u003e. Results were calculated using the percent input method. The acquired \u0026Delta;Ct values were used to assess statistical significance between treatments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are displayed as mean \u0026plusmn; standard deviation (SD) or mean \u0026plusmn; standard error of mean (SEM). Statistical analyses were conducted using GraphPad Prism 11. Where appropriate, Student\u0026rsquo;s t-test or one-way ANOVA followed by Tukey HSD post-Hoc analysis was used to assess statistical significance amongst treatments. Values with P \u0026le; 0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the conclusions of this article are included in the article, its supplementary files or available from the corresponding author under reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGO, MP and SS performed experiments. GO, MP and SEL designed experiments and analyzed data. JP provided expertise and guidance relating to generation and validation of NT and ATF6 knockout cells. GO and SEL prepared the manuscript. SEL devised the study, acquired funding and oversaw the research program. All listed authors reviewed the manuscript and provided critical feedback.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure and competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Canada Research Chairs Program (CRC-2018-00305), NSERC Discovery Grant (RGPIN-2020-04896), Research Manitoba and CFI JELF awards to SEL. GO was supported by a Canada Graduate Doctoral Scholarship (CGSD-588623-2024). We would like to acknowledge the support provided by the QuIPS Platform funded by the CancerCare Manitoba Foundation in aiding the generation of knockout cell lines required for this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCox JS, Shamu CE, Walter P. 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The UPR\u0026rsquo;s primary function is to help cells manage and resolve ER stress. Compared with the well-characterized IRE1 and PERK pathways, how ATF6 shapes the wider UPR network is still largely unresolved. Using pharmacological inhibition, genetic knockout, and inducible expression models, we show that ATF6 signaling intersects with both the PERK and IRE1 branches of the UPR. During early ER stress, ATF6 promotes PERK expression, with inhibition or loss of ATF6 lowering PERK levels, while selective induction of active ATF6 drives PERK upregulation. As stress shifts from acute to prolonged exposure, ATF6 signaling helps to dampen IRE1 RNase activity. Cells lacking ATF6 or treated with ATF6 inhibitors exhibit prolonged IRE1 RNase activity, while induction of active ATF6 suppresses IRE1 signaling. Our findings identify an unappreciated role for ATF6 as a temporal modulator of UPR signaling, underscoring the importance of communication between ER stress sensors in fine-tuning adaptive responses that dictate cellular outcomes during ER stress.\u003c/p\u003e","manuscriptTitle":"ATF6 shapes PERK and IRE1 signaling dynamics during ER stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-21 16:18:58","doi":"10.21203/rs.3.rs-9373070/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-04-27T09:13:48+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-04-27T03:53:03+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-04-23T15:26:41+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-04-13T10:16:52+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-04-13T09:55:38+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-04-13T09:30:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-10T08:49:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-10T00:47:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2026-04-10T00:47:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7b5fc7e5-1cd3-42bb-a38f-7e848184bf0a","owner":[],"postedDate":"April 21st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":66068863,"name":"Biological sciences/Cell biology/Cell signalling/Stress signalling"},{"id":66068864,"name":"Biological sciences/Cell biology/Proteolysis/Protein quality control"},{"id":66068865,"name":"Biological sciences/Cell biology/Organelles/Endoplasmic reticulum"}],"tags":[],"updatedAt":"2026-04-27T09:19:50+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-21 16:18:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9373070","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9373070","identity":"rs-9373070","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}