{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,3]],"date-time":"2026-10-03T03:13:30Z","timestamp":1790997210756,"version":"4.1.0"},"reference-count":53,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2023,3,31]],"date-time":"2023-03-31T00:00:00Z","timestamp":1680220800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ministry for Innovation and Technology in Hungary","award":["RRF-2.3.1-21-2022-00012"],"award-info":[{"award-number":["RRF-2.3.1-21-2022-00012"]}]},{"name":"\u201cNational Laboratory on Human Reproduction\u201d program","award":["RRF-2.3.1-21-2022-00012"],"award-info":[{"award-number":["RRF-2.3.1-21-2022-00012"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Instead of Western blot being considered as a gold standard for intracellular protein expression assays, we developed a novel multiplexed high throughput (180 tests\/day) in situ manual protein expression method directly in 96-well plates using 25,000\u2013100,000 cells\/well after formaldehyde fixation and Triton X 100 permeabilization. HepG2 cells were treated with ochratoxin A (OTA) and staurosporine (STP) to induce apoptosis. Antioxidant and apoptotic cell signaling protein expression were studied by various rabbit primary antibodies and HRP labeled secondary antibodies. The HRP labeled immune complexes were developed by H2O2\/Ampliflu Red fluorogenic reagent and measured in a plate reader. Our assay can simultaneously quantify 22 protein antigens in one plate with 4 technical replicates with an interassay imprecision of &lt;10% CV. The fluorescence signals are referred to total intracellular protein contents in the wells and given as fluorescence\/protein ratio FPR, expressed as % of the controls (FPR %). OTA caused a dose\u2013response increase (p &lt; 0.05\u2013p &lt; 0.001) in SOD2, CAT, ALB, CASP3,7,9, BCL2, BAX, Nf-kB, phospho-Erk1\/2\/Erk1\/2, phospho-Akt\/Akt, phospho-p38\/p38, and phospho-PPARg\/PPARg levels while phospho-AMPK\/AMPK ratios decreased (p &lt; 0.05\u2013p &lt; 0.001). On the contrary, STP induced a dose\u2013response decrease (p &lt; 0.05\u2013p &lt; 0.001) in CASP3,7,9, BAX, BCL2, Nf-kB and phospho-Erk1\/2\/Erk1\/2 expression while B-ACT, phospho-Akt\/Akt, phospho-p38\/p38 and phospho-PPARg\/PPARg ratios increased.<\/jats:p>","DOI":"10.3390\/ijms24076564","type":"journal-article","created":{"date-parts":[[2023,3,31]],"date-time":"2023-03-31T10:19:33Z","timestamp":1680257973000},"page":"6564","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Multiplexed Fluorescence Plate Reader In Situ Protein Expression Assay in Apoptotic HepG2 Cells"],"prefix":"10.3390","volume":"24","author":[{"given":"Rita","family":"Jakabfi-Csepregi","sequence":"first","affiliation":[{"name":"Department of Laboratory Medicine, Medical School, University of P\u00e9cs, 7624 P\u00e9cs, Hungary"},{"name":"Szent\u00e1gothai Research Center, University of P\u00e9cs, 7624 P\u00e9cs, Hungary"},{"name":"Hungarian National Laboratory on Reproduction, University of P\u00e9cs, 7624 P\u00e9cs, Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5298-5401","authenticated-orcid":false,"given":"G\u00e1bor L.","family":"Kov\u00e1cs","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, Medical School, University of P\u00e9cs, 7624 P\u00e9cs, Hungary"},{"name":"Szent\u00e1gothai Research Center, University of P\u00e9cs, 7624 P\u00e9cs, Hungary"},{"name":"Hungarian National Laboratory on Reproduction, University of P\u00e9cs, 7624 P\u00e9cs, Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0151-4682","authenticated-orcid":false,"given":"P\u00e9ter","family":"Kaltenecker","sequence":"additional","affiliation":[{"name":"Szent\u00e1gothai Research Center, University of P\u00e9cs, 7624 P\u00e9cs, Hungary"},{"name":"Hungarian National Laboratory on Reproduction, University of P\u00e9cs, 7624 P\u00e9cs, Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3910-5745","authenticated-orcid":false,"given":"Tam\u00e1s","family":"K\u0151szegi","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, Medical School, University of P\u00e9cs, 7624 P\u00e9cs, Hungary"},{"name":"Szent\u00e1gothai Research Center, University of P\u00e9cs, 7624 P\u00e9cs, Hungary"},{"name":"Hungarian National Laboratory on Reproduction, University of P\u00e9cs, 7624 P\u00e9cs, Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,3,31]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"222","DOI":"10.1038\/srep00222","article-title":"Accurate RT-qPCR gene expression analysis on cell culture lysates","volume":"2","author":"Mestdagh","year":"2012","journal-title":"Sci. Rep."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"113608","DOI":"10.1016\/j.ab.2020.113608","article-title":"A systematic approach to quantitative Western blot analysis","volume":"593","author":"Harford","year":"2020","journal-title":"Anal. Biochem."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"13929","DOI":"10.1002\/anie.201906920","article-title":"In Situ Single-Cell Western Blot on Adherent Cell Culture","volume":"58","author":"Zhang","year":"2019","journal-title":"Angew Chem. Int. Ed. Engl."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"749","DOI":"10.1038\/nmeth.2992","article-title":"Single-cell western blotting","volume":"11","author":"Hughes","year":"2014","journal-title":"Nat. Methods"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"630","DOI":"10.1038\/s41576-020-0258-4","article-title":"mRNAs, proteins and the emerging principles of gene expression control","volume":"21","author":"Buccitelli","year":"2020","journal-title":"Nat. Rev. Genet."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"535","DOI":"10.1016\/j.cell.2016.03.014","article-title":"On the Dependency of Cellular Protein Levels on mRNA Abundance","volume":"165","author":"Liu","year":"2016","journal-title":"Cell"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"463813","DOI":"10.1016\/j.chroma.2023.463813","article-title":"The development of microfluidic-based western blotting: Technical advances and future perspectives","volume":"1691","author":"Desire","year":"2023","journal-title":"J. Chromatogr. A"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1016\/j.cbpa.2013.11.012","article-title":"Cross-reactivity in antibody microarrays and multiplexed sandwich assays: Shedding light on the dark side of multiplexing","volume":"18","author":"Juncker","year":"2014","journal-title":"Curr. Opin. Chem. Biol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1186\/s12936-020-03225-5","article-title":"Comparison of ELISA with electro-chemiluminescence technology for the qualitative and quantitative assessment of serological responses to vaccination","volume":"19","author":"Bolton","year":"2020","journal-title":"Malar. J."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1016\/0003-2697(81)90281-5","article-title":"\"Western blotting\": Electrophoretic transfer of proteins from sodium dodecyl sulfate\u2014Polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A","volume":"112","author":"Burnette","year":"1981","journal-title":"Anal. Biochem."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"581","DOI":"10.1002\/elps.1150110709","article-title":"Blotting efficiency investigated by using two-dimensional electrophoresis, hydrophobic membranes and proteins from different sources","volume":"11","author":"Jungblut","year":"1990","journal-title":"Electrophoresis"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1844","DOI":"10.1038\/nprot.2010.142","article-title":"Automated microfluidic protein immunoblotting","volume":"5","author":"He","year":"2010","journal-title":"Nat. Protoc."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1148","DOI":"10.1002\/elps.201200534","article-title":"A novel method that improves sensitivity of protein detection in PAGE and Western blot","volume":"34","author":"Marciano","year":"2013","journal-title":"Electrophoresis"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"6703","DOI":"10.1021\/acs.analchem.6b00705","article-title":"Multiplexed Western Blotting Using Microchip Electrophoresis","volume":"88","author":"Jin","year":"2016","journal-title":"Anal. Chem."},{"key":"ref_15","first-page":"7002","article-title":"Recent Advances in Microscale Western Blotting","volume":"8","author":"Sanders","year":"2016","journal-title":"Anal. Methods Adv. Methods Appl."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"512","DOI":"10.1101\/pdb.prot102251","article-title":"Analysis of Proteins by Immunoblotting","volume":"2021","author":"Kielkopf","year":"2021","journal-title":"Cold Spring Harb. Protoc."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1309","DOI":"10.1007\/s10096-021-04203-8","article-title":"Automated Western immunoblotting detection of anti-SARS-CoV-2 serum antibodies","volume":"40","author":"Edouard","year":"2021","journal-title":"Eur. J. Clin. Microbiol. Infect. Dis."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"429","DOI":"10.4103\/1947-2714.100998","article-title":"Western blot: Technique, theory, and trouble shooting","volume":"4","author":"Mahmood","year":"2012","journal-title":"North Am. J. Med. Sci."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1183","DOI":"10.1080\/14737159.2020.1857734","article-title":"Protein array-based companion diagnostics in precision medicine","volume":"20","author":"Poulsen","year":"2020","journal-title":"Expert Rev. Mol. Diagn."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"154","DOI":"10.1080\/15321819.2020.1838924","article-title":"ELISA versus LUMINEX assay for measuring mouse metabolic hormones and cytokines: Sharing the lessons I have learned","volume":"42","author":"Mountjoy","year":"2021","journal-title":"J. Immunoass. Immunochem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"167","DOI":"10.1007\/978-1-4939-7346-0_10","article-title":"Multiparametric Analysis of Apoptosis by Flow Cytometry","volume":"1678","author":"Telford","year":"2018","journal-title":"Methods Mol. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"393","DOI":"10.1007\/s00204-016-1677-z","article-title":"Comparative analysis of 3D culture methods on human HepG2 cells","volume":"91","author":"Luckert","year":"2017","journal-title":"Arch. Toxicol."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Koszegi, T., and Poor, M. (2016). Ochratoxin A: Molecular Interactions, Mechanisms of Toxicity and Prevention at the Molecular Level. Toxins, 8.","DOI":"10.3390\/toxins8040111"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Garcia-Perez, E., Ryu, D., Lee, C., and Lee, H.J. (2021). Ochratoxin A Induces Oxidative Stress in HepG2 Cells by Impairing the Gene Expression of Antioxidant Enzymes. Toxins, 13.","DOI":"10.3390\/toxins13040271"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1016\/j.vascn.2016.01.004","article-title":"Multiparametric luminescent cell viability assay in toxicology models: A critical evaluation","volume":"79","author":"Sali","year":"2016","journal-title":"J. Pharm. Toxicol. Methods"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/j.tox.2016.09.001","article-title":"Ochratoxin A activates neutrophils and kills these cells through necrosis, an effect eliminated through its conversion into ochratoxin alpha","volume":"368\u2013369","author":"Kupski","year":"2016","journal-title":"Toxicology"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"766","DOI":"10.1016\/j.fct.2013.05.043","article-title":"A review on ochratoxin A transcriptomic studies","volume":"59","author":"Vettorazzi","year":"2013","journal-title":"Food Chem. Toxicol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1016\/j.mrfmmm.2014.05.001","article-title":"DNA damage and S phase arrest induced by Ochratoxin A in human embryonic kidney cells (HEK 293)","volume":"765","author":"Yang","year":"2014","journal-title":"Mutat. Res."},{"key":"ref_29","first-page":"565","article-title":"Staurosporine-induced apoptosis in Chang liver cells is associated with down-regulation of Bcl-2 and Bcl-XL","volume":"13","author":"Giuliano","year":"2004","journal-title":"Int. J. Mol. Med."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1186\/s40001-019-0365-x","article-title":"Staurosporine induces apoptosis in pancreatic carcinoma cells PaTu 8988t and Panc-1 via the intrinsic signaling pathway","volume":"24","author":"Malsy","year":"2019","journal-title":"Eur. J. Med. Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1038\/ja.2008.4","article-title":"Chemical biology of natural indolocarbazole products: 30 years since the discovery of staurosporine","volume":"62","author":"Nakano","year":"2009","journal-title":"J. Antibiot."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"5691","DOI":"10.1039\/D1CS00991E","article-title":"Dissecting the role of protein phosphorylation: A chemical biology toolbox","volume":"51","author":"Bilbrough","year":"2022","journal-title":"Chem. Soc. Rev."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1038\/nbt1358","article-title":"A quantitative analysis of kinase inhibitor selectivity","volume":"26","author":"Karaman","year":"2008","journal-title":"Nat. Biotechnol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"3250","DOI":"10.1096\/fj.10-177527","article-title":"Triggering of a novel intrinsic apoptosis pathway by the kinase inhibitor staurosporine: Activation of caspase-9 in the absence of Apaf-1","volume":"25","author":"Manns","year":"2011","journal-title":"FASEB J."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1016\/S0006-8993(02)02365-X","article-title":"Release of cytochrome c into the extracellular space contributes to neuronal apoptosis induced by staurosporine","volume":"934","author":"Ahlemeyer","year":"2002","journal-title":"Brain Res."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1016\/S0009-2797(02)00078-9","article-title":"Interaction of ochratoxin A with human serum albumin. Binding sites localized by competitive interactions with the native protein and its recombinant fragments","volume":"141","author":"Perry","year":"2002","journal-title":"Chem. Biol. Interact."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Faisal, Z., Derdak, D., Lemli, B., Kunsagi-Mate, S., Balint, M., Hetenyi, C., Csepregi, R., Koszegi, T., Sueck, F., and Cramer, B. (2018). Interaction of 2\u2019R-ochratoxin A with Serum Albumins: Binding Site, Effects of Site Markers, Thermodynamics, Species Differences of Albumin-binding, and Influence of Albumin on Its Toxicity in MDCK Cells. Toxins, 10.","DOI":"10.3390\/toxins10090353"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"2123","DOI":"10.1016\/S0024-3205(01)01296-6","article-title":"Characterization of ochratoxin A transport by human organic anion transporters","volume":"69","author":"Jung","year":"2001","journal-title":"Life Sci."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1381","DOI":"10.3390\/toxins2061381","article-title":"Molecular mechanism of ochratoxin a transport in the kidney","volume":"2","author":"Anzai","year":"2010","journal-title":"Toxins"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Erceg, S., Mateo, E.M., Zipancic, I., Rodriguez Jimenez, F.J., Perez Arago, M.A., Jimenez, M., Soria, J.M., and Garcia-Esparza, M.A. (2019). Assessment of Toxic Effects of Ochratoxin A in Human Embryonic Stem Cells. Toxins, 11.","DOI":"10.3390\/toxins11040217"},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Yang, X., Gao, Y., Yan, Q., Bao, X., Zhao, S., Wang, J., and Zheng, N. (2019). Transcriptome Analysis of Ochratoxin A-Induced Apoptosis in Differentiated Caco-2 Cells. Toxins, 12.","DOI":"10.3390\/toxins12010023"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1352","DOI":"10.1016\/j.immuni.2019.05.020","article-title":"Caspases in Cell Death, Inflammation, and Disease","volume":"50","author":"Lamkanfi","year":"2019","journal-title":"Immunity"},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Banjara, S., Suraweera, C.D., Hinds, M.G., and Kvansakul, M. (2020). The Bcl-2 Family: Ancient Origins, Conserved Structures, and Divergent Mechanisms. Biomolecules, 10.","DOI":"10.3390\/biom10010128"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"175","DOI":"10.1038\/s41580-018-0089-8","article-title":"Regulation of apoptosis in health and disease: The balancing act of BCL-2 family proteins","volume":"20","author":"Singh","year":"2019","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1038\/nrm3722","article-title":"Control of apoptosis by the BCL-2 protein family: Implications for physiology and therapy","volume":"15","author":"Czabotar","year":"2014","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"718","DOI":"10.1016\/j.chemosphere.2018.02.009","article-title":"Immunotoxicity of ochratoxin A and aflatoxin B1 in combination is associated with the nuclear factor kappa B signaling pathway in 3D4\/21 cells","volume":"199","author":"Hou","year":"2018","journal-title":"Chemosphere"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1186\/2110-5820-3-4","article-title":"Specific antioxidant properties of human serum albumin","volume":"3","author":"Taverna","year":"2013","journal-title":"Ann. Intensive Care"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"271","DOI":"10.3892\/ijmm.2017.3036","article-title":"The crucial role of protein phosphorylation in cell signaling and its use as targeted therapy (Review)","volume":"40","author":"Ardito","year":"2017","journal-title":"Int. J. Mol. Med."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1313","DOI":"10.1007\/s00204-014-1311-x","article-title":"Ochratoxin A activates opposing c-MET\/PI3K\/Akt and MAPK\/ERK 1-2 pathways in human proximal tubule HK-2 cells","volume":"89","author":"Ozcan","year":"2015","journal-title":"Arch. Toxicol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"796","DOI":"10.1038\/nsb1097-796","article-title":"Protein kinase inhibition by staurosporine revealed in details of the molecular interaction with CDK2","volume":"4","author":"Lawrie","year":"1997","journal-title":"Nat. Struct. Biol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"770","DOI":"10.1038\/35037710","article-title":"The biochemistry of apoptosis","volume":"407","author":"Hengartner","year":"2000","journal-title":"Nature"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"489","DOI":"10.1039\/b923848b","article-title":"Protein kinase inhibition of clinically important staurosporine analogues","volume":"27","author":"Gani","year":"2010","journal-title":"Nat. Prod. Rep."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"1112","DOI":"10.1038\/nprot.2006.179","article-title":"Sulforhodamine B colorimetric assay for cytotoxicity screening","volume":"1","author":"Vichai","year":"2006","journal-title":"Nat. Protoc."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/7\/6564\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T19:07:46Z","timestamp":1760123266000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/7\/6564"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,3,31]]},"references-count":53,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2023,4]]}},"alternative-id":["ijms24076564"],"URL":"https:\/\/doi.org\/10.3390\/ijms24076564","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,3,31]]}}}