{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T14:32:18Z","timestamp":1787149938221,"version":"build-2736575974"},"reference-count":49,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T00:00:00Z","timestamp":1787097600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100006769","name":"Russian Science Foundation","doi-asserted-by":"publisher","award":["25-24-00140"],"award-info":[{"award-number":["25-24-00140"]}],"id":[{"id":"10.13039\/501100006769","id-type":"DOI","asserted-by":"publisher"}]},{"award":["25-24-00140"],"award-info":[{"award-number":["25-24-00140"]}],"id":[{"id":"https:\/\/ror.org\/03y2gwe85","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Int. J. Mol. Sci.","IJMS"],"abstract":"<jats:p>Chinese hamster ovary (CHO) producer cells differ in their capacity to accommodate secretory and endoplasmic reticulum (ER) stress, but how producer and host-cell backgrounds are associated with ER-proteostasis responses remains poorly understood. We compared 24 h dithiothreitol (DTT)-induced reductive ER stress responses in two clonal producers: CHO-S-derived HB8 secreting human chorionic gonadotropin and apoptosis-resistant CHO-4BGD-derived DUL secreting a GLP-1\u2013Fc fusion protein with similar specific productivities. Responses were analyzed by strand-specific RNA-seq, Xbp1-splicing RT-PCR, immunoblotting, functional enrichment, and curated-module analysis. Analysis of the complete three-replicate dataset identified 299 DTT-responsive genes in HB8 and 877 in DUL, demonstrating a broader transcriptional response in the DUL clone. Analysis after exclusion of the atypical HB8#3 matched pair yielded 404 and 1019 differentially expressed genes, respectively, and was used for detailed sensitivity analysis. Both producers showed ISR\/ATF4\/CHOP-associated transcriptional activation and Xbp1 mRNA splicing, whereas changes in total Xbp1 and Hspa5\/BiP transcript abundance were limited. BiP and CHOP accumulation was detected at the protein level, while phospho-eIF2\u03b1 and ATF6 responses were variable. Baseline RNA-seq data comparison revealed extensive transcriptional divergence between HB8 and DUL. HB8 showed higher expression of several classical ER folding\/redox factors, whereas DUL showed higher expression of selected ISR-, quality-control- and stress-survival-associated genes. DUL additionally displayed broader vesicle\/endocytic and amino-acid\/glutathione-related remodeling. Thus, the producers occupy distinct ER-proteostasis states, and DUL mounts a broader, but not uniformly stronger, canonical UPR response to reductive ER stress.<\/jats:p>","DOI":"10.3390\/ijms27167427","type":"journal-article","created":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T14:07:34Z","timestamp":1787148454000},"page":"7427","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["DTT-Induced Transcriptome Profiling Reveals Divergent ER Proteostasis and Stress-Adaptation Programs in CHO-S\/HB8 and CHO-4BGD\/DUL Producer Clones"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9483-2892","authenticated-orcid":false,"given":"Nadezhda A.","family":"Orlova","sequence":"first","affiliation":[{"name":"Laboratory of Mammalian Cell Bioengineering, Skryabin Institute of Bioengineering, Federal Research Centre \u2018Fundamentals of Biotechnology\u2019 of the Russian Academy of Sciences, 60 let Oktjabrja pr-t 7, bld. 1, 117312 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nadezhda A.","family":"Potapova","sequence":"additional","affiliation":[{"name":"Association \u201cTechnological Platform BioTech 2030\u201d, Leninsky Prospekt, 33, Bld. 3, 119071 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-5118-5067","authenticated-orcid":false,"given":"Rolan R.","family":"Shaifutdinov","sequence":"additional","affiliation":[{"name":"Laboratory of Mammalian Cell Bioengineering, Skryabin Institute of Bioengineering, Federal Research Centre \u2018Fundamentals of Biotechnology\u2019 of the Russian Academy of Sciences, 60 let Oktjabrja pr-t 7, bld. 1, 117312 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0064-4458","authenticated-orcid":false,"given":"Ivan I.","family":"Vorobiev","sequence":"additional","affiliation":[{"name":"Laboratory of Mammalian Cell Bioengineering, Skryabin Institute of Bioengineering, Federal Research Centre \u2018Fundamentals of Biotechnology\u2019 of the Russian Academy of Sciences, 60 let Oktjabrja pr-t 7, bld. 1, 117312 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"628","DOI":"10.1080\/07388551.2022.2047004","article-title":"The secretory pathway\u2014The key for unlocking the potential of Chinese hamster ovary cell factories for manufacturing therapeutic proteins","volume":"43","author":"Torres","year":"2023","journal-title":"Crit. Rev. Biotechnol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1016\/j.ymben.2022.03.009","article-title":"Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins","volume":"72","author":"Malm","year":"2022","journal-title":"Metab. Eng."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Sebastiao, M.J., Hoffman, M., Escandell, J., Tousi, F., Zhang, J., Figueroa, B., DeMaria, C., and Gomes-Alves, P. (2023). Identification of Mispairing Omic Signatures in Chinese Hamster Ovary (CHO) Cells Producing a Tri-Specific Antibody. Biomedicines, 11.","DOI":"10.3390\/biomedicines11112890"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1016\/j.csbj.2024.12.006","article-title":"Identification of cellular signatures associated with chinese hamster ovary cell adaptation for secretion of antibodies","volume":"27","author":"Bai","year":"2025","journal-title":"Comput. Struct. Biotechnol. J."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1099","DOI":"10.1016\/S1097-2765(00)00108-8","article-title":"Regulated translation initiation controls stress-induced gene expression in mammalian cells","volume":"6","author":"Harding","year":"2000","journal-title":"Mol. Cell"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"881","DOI":"10.1016\/S0092-8674(01)00611-0","article-title":"XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor","volume":"107","author":"Yoshida","year":"2001","journal-title":"Cell"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"3787","DOI":"10.1091\/mbc.10.11.3787","article-title":"Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress","volume":"10","author":"Haze","year":"1999","journal-title":"Mol. Biol. Cell"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1186\/s12964-019-0499-z","article-title":"Cell death induced by the ER stressor thapsigargin involves death receptor 5, a non-autophagic function of MAP1LC3B, and distinct contributions from unfolded protein response components","volume":"18","author":"Lindner","year":"2020","journal-title":"Cell Commun. Signal."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2537","DOI":"10.1091\/mbc.e03-09-0693","article-title":"Activation of mammalian unfolded protein response is compatible with the quality control system operating in the endoplasmic reticulum","volume":"15","author":"Nadanaka","year":"2004","journal-title":"Mol. Biol. Cell"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"16620","DOI":"10.1038\/s41598-020-73573-4","article-title":"Perfusion reduces bispecific antibody aggregation via mitigating mitochondrial dysfunction-induced glutathione oxidation and ER stress in CHO cells","volume":"10","author":"Sinharoy","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"e1800352","DOI":"10.1002\/biot.201800352","article-title":"Multi-Omics Study on the Impact of Cysteine Feed Level on Cell Viability and mAb Production in a CHO Bioprocess","volume":"14","author":"Ali","year":"2019","journal-title":"Biotechnol. J."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"e2200638","DOI":"10.1002\/biot.202200638","article-title":"Cystine and tyrosine feed reduces oxidative and ER stress in CHO cells","volume":"18","author":"Shibafuji","year":"2023","journal-title":"Biotechnol. J."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"14141","DOI":"10.1038\/s41598-024-64767-1","article-title":"RNASeq highlights ATF6 pathway regulators for CHO cell engineering with different impacts of ATF6beta and WFS1 knockdown on fed-batch production of IgG(1)","volume":"14","author":"Rives","year":"2024","journal-title":"Sci. Rep."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"391","DOI":"10.2174\/1389201023666220603121316","article-title":"Factors and Mechanisms Affecting the Secretion of Recombinant Protein in CHO Cells","volume":"24","author":"Yang","year":"2023","journal-title":"Curr. Pharm. Biotechnol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"697","DOI":"10.1002\/btpr.1693","article-title":"A CHO cell line engineered to express XBP1 and ERO1-Lalpha has increased levels of transient protein expression","volume":"29","author":"Cain","year":"2013","journal-title":"Biotechnol. Prog."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/j.ymben.2012.12.003","article-title":"CHO cell engineering to prevent polypeptide aggregation and improve therapeutic protein secretion","volume":"21","author":"Girod","year":"2014","journal-title":"Metab. Eng."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Orlova, N.A., Sinegubova, M.V., Kolesov, D.E., Khodak, Y.A., Tatarskiy, V.V., and Vorobiev, I.I. (2025). Genomic and Phenotypic Characterization of CHO 4BGD Cells with Quad Knockout and Overexpression of Two Housekeeping Genes That Allow for Metabolic Selection and Extended Fed-Batch Culturing. Cells, 14.","DOI":"10.3390\/cells14100692"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Shaifutdinov, R.R., Sinegubova, M.V., Vorobiev, I.I., Prokhorova, P.E., Podkorytov, A.B., and Orlova, N.A. (2025). Production of a Dulaglutide Analogue by Apoptosis-Resistant Chinese Hamster Ovary Cells in a 3-Week Fed-Batch Process. Pharmaceuticals, 18.","DOI":"10.3390\/ph18121896"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"20598","DOI":"10.1016\/S0021-9258(20)80767-4","article-title":"The secretory pathway is normal in dithiothreitol-treated cells, but disulfide-bonded proteins are reduced and reversibly retained in the endoplasmic reticulum","volume":"268","author":"Lodish","year":"1993","journal-title":"J. Biol. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"847","DOI":"10.1038\/cdd.2011.165","article-title":"Mutation of ATF4 mediates resistance of neuronal cell lines against oxidative stress by inducing xCT expression","volume":"19","author":"Lewerenz","year":"2012","journal-title":"Cell Death Differ."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1095","DOI":"10.1038\/embor.2012.156","article-title":"Mammalian proapoptotic factor ChaC1 and its homologues function as \u03b3-glutamyl cyclotransferases acting specifically on glutathione","volume":"13","author":"Kumar","year":"2012","journal-title":"EMBO Rep."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1243","DOI":"10.1038\/sj.emboj.7600596","article-title":"TRB3, a novel ER stress-inducible gene, is induced via ATF4\u2013CHOP pathway and is involved in cell death","volume":"24","author":"Ohoka","year":"2005","journal-title":"EMBO J."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Yoon, S.B., Park, Y.H., Choi, S.A., Yang, H.J., Jeong, P.S., Cha, J.J., Lee, S., Lee, S.H., Lee, J.H., and Sim, B.W. (2019). Real-time PCR quantification of spliced X-box binding protein 1 (XBP1) using a universal primer method. PLoS ONE, 14.","DOI":"10.1371\/journal.pone.0219978"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2443","DOI":"10.1242\/jcs.115.11.2443","article-title":"BiP is feed-back regulated by control of protein translation efficiency","volume":"115","author":"Bienert","year":"2002","journal-title":"J. Cell Sci."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"aad3867","DOI":"10.1126\/science.aad3867","article-title":"Translation from the 5\u2032 untranslated region shapes the integrated stress response","volume":"351","author":"Starck","year":"2016","journal-title":"Science"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Herath, V., and Verchot, J. (2022). Comprehensive transcriptome analysis reveals genome-wide changes associated with endoplasmic reticulum (ER) stress in potato (Solanum tuberosum L.). Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms232213795"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"897","DOI":"10.1016\/S1097-2765(00)80330-5","article-title":"PERK is essential for translational regulation and cell survival during the unfolded protein response","volume":"5","author":"Harding","year":"2000","journal-title":"Mol. Cell"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"C121","DOI":"10.1152\/ajpcell.1999.277.1.C121","article-title":"Disruption of disulfide bonds exhibits differential effects on trafficking of regulated secretory proteins","volume":"277","author":"Gorr","year":"1999","journal-title":"Am. J. Physiol. Cell Physiol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"15878","DOI":"10.1074\/jbc.M114.635144","article-title":"Human CHAC1 protein degrades glutathione, and mRNA induction is regulated by the transcription factors ATF4 and ATF3 and a bipartite ATF\/CRE regulatory element","volume":"290","author":"Crawford","year":"2015","journal-title":"J. Biol. Chem."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"e1900565","DOI":"10.1002\/biot.201900565","article-title":"Multi-omics reveals impact of cysteine feed concentration and resulting redox imbalance on cellular energy metabolism and specific productivity in CHO cell bioprocessing","volume":"15","author":"Ali","year":"2020","journal-title":"Biotechnol. J."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"e18908","DOI":"10.7717\/peerj.18908","article-title":"Increased glycoprotein hormone yield in stably transfected CHO cells using human serum albumin signal peptide for beta-chains","volume":"13","author":"Sinegubova","year":"2025","journal-title":"PeerJ"},{"key":"ref_32","unstructured":"Vorobiev, I.I., Orlova, N.A., Sinegubova, M.V., Klishin, A.A., and Zyryanov, D.A. (2024). Plasmid for Expression of Recombinant Human Chorionic Gonadotropin (hCG), Plasmid for Expression of Recombinant Alpha- and Beta-Subunits of hCG, Monoclonal Mammalian Cell Line Producing hCG, Method for Producing Recombinant hCG. (RU 2834784), Russian Patent."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"907","DOI":"10.1038\/s41587-019-0201-4","article-title":"Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype","volume":"37","author":"Kim","year":"2019","journal-title":"Nat. Biotechnol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2078","DOI":"10.1093\/bioinformatics\/btp352","article-title":"The Sequence Alignment\/Map format and SAMtools","volume":"25","author":"Li","year":"2009","journal-title":"Bioinformatics"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"923","DOI":"10.1093\/bioinformatics\/btt656","article-title":"featureCounts: An efficient general purpose program for assigning sequence reads to genomic features","volume":"30","author":"Liao","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"550","DOI":"10.1186\/s13059-014-0550-8","article-title":"Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2","volume":"15","author":"Love","year":"2014","journal-title":"Genome Biol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"2628","DOI":"10.1093\/bioinformatics\/btz931","article-title":"ShinyGO: A graphical gene-set enrichment tool for animals and plants","volume":"36","author":"Ge","year":"2020","journal-title":"Bioinformatics"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"417","DOI":"10.1038\/nmeth.4197","article-title":"Salmon provides fast and bias-aware quantification of transcript expression","volume":"14","author":"Patro","year":"2017","journal-title":"Nat. Methods"},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Read, A., and Schr\u00f6der, M. (2021). The Unfolded Protein Response: An Overview. Biology, 10.","DOI":"10.3390\/biology10050384"},{"key":"ref_40","unstructured":"Reactome (2026, August 12). ATF4 Activates Genes in Response to Endoplasmic Reticulum Stress. Available online: https:\/\/reactome.org\/content\/detail\/R-HSA-380994."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"jcs232850","DOI":"10.1242\/jcs.232850","article-title":"ER-Associated Degradation in Health and Disease\u2014From Substrate to Organism","volume":"132","author":"Bhattacharya","year":"2019","journal-title":"J. Cell Sci."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"a041250","DOI":"10.1101\/cshperspect.a041250","article-title":"Mechanism of Protein Translocation by the Sec61 Translocon Complex","volume":"15","author":"Itskanov","year":"2023","journal-title":"Cold Spring Harb. Perspect. Biol."},{"key":"ref_43","unstructured":"Reactome (2026, August 12). Asparagine N-Linked Glycosylation. Available online: https:\/\/reactome.org\/content\/detail\/R-HSA-446203."},{"key":"ref_44","unstructured":"Reactome (2026, August 12). COPII-Mediated Vesicle Transport. Available online: https:\/\/reactome.org\/content\/detail\/R-HSA-204005."},{"key":"ref_45","unstructured":"Reactome (2026, August 12). ER to Golgi Anterograde Transport. Available online: https:\/\/reactome.org\/content\/detail\/R-HSA-199977."},{"key":"ref_46","unstructured":"Reactome (2026, August 12). COPI-Dependent Golgi-to-ER Retrograde Traffic. Available online: https:\/\/reactome.org\/content\/detail\/R-HSA-6811434."},{"key":"ref_47","unstructured":"(2026, August 12). Autophagy. Available online: https:\/\/reactome.org\/content\/detail\/R-HSA-9612973."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"107525","DOI":"10.1016\/j.pharmthera.2020.107525","article-title":"Role of the ERO1\u2013PDI Interaction in Oxidative Protein Folding and Disease","volume":"210","author":"Shergalis","year":"2020","journal-title":"Pharmacol. Ther."},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Glin\u0161ek, K., Bozovi\u010dar, K., and Bratkovi\u010d, T. (2023). CRISPR Technologies in Chinese Hamster Ovary Cell Line Engineering. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24098144"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/16\/7427\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T14:08:45Z","timestamp":1787148525000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/16\/7427"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,19]]},"references-count":49,"journal-issue":{"issue":"16","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["ijms27167427"],"URL":"https:\/\/doi.org\/10.3390\/ijms27167427","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,19]]}}}