{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T03:02:31Z","timestamp":1789441351066,"version":"build-2803163510"},"reference-count":10,"publisher":"Wiley","issue":"3","license":[{"start":{"date-parts":[[2026,3,19]],"date-time":"2026-03-19T00:00:00Z","timestamp":1773878400000},"content-version":"vor","delay-in-days":18,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"},{"start":{"date-parts":[[2026,3,1]],"date-time":"2026-03-01T00:00:00Z","timestamp":1772323200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2026,3]]},"abstract":"Abstract<\/jats:title>\n \n Reactive oxygen species (ROS) are highly reactive oxygen\u2010based molecules\u00a0comprising hydrogen peroxide, hydroxyl radicals, superoxide anion, and singlet oxygen. These species are produced intracellularly and play an important role in cellular signaling and metabolism. Their high reactivity damages intracellular macromolecules such as lipids and DNA. In cancer biology, ROS display a dual role: they promote cancer cell proliferation at low to moderate levels, whereas excessive accumulation overwhelms antioxidant defenses, causing oxidative stress and apoptosis. This has resulted in therapeutic strategies that selectively increase ROS in cancer cells to induce apoptosis. Vitamin D has demonstrated anti\u2010cancer properties, with one proposed mechanism involving ROS\u2010mediated apoptosis. This article outlines a workflow to investigate ROS\u2010induced cellular damage by vitamin D\n 3<\/jats:sub>\n in HeLa cervical cancer cells. The study begins with quantification of ROS levels and assessment of mitochondrial membrane potential in HeLa cultures. Transmission electron microscopy is used to examine mitochondrial ultrastructure. Lipid peroxidation quantifies downstream ROS\u2010mediated membrane and cellular injury. Antioxidant enzyme activities, including superoxide dismutase and catalase, measure cellular anti\u2010oxidative defence capacity. Lastly, the role of ROS inhibition of AKT signaling, leading to reduced cell survival and apoptosis, is quantified by immunoblotting. \u00a9 2026 The Author(s).\n Current Protocols<\/jats:italic>\n published by Wiley Periodicals LLC.\n <\/jats:p>\n \n Basic Protocol 1<\/jats:bold>\n : Preparation of HeLa cell cultures and treatment with vitamin D\n 3<\/jats:sub>\n <\/jats:p>\n \n Basic Protocol 2<\/jats:bold>\n : Quantification of ROS\u2010positive cells using the Muse Oxidative Stress assay\n <\/jats:p>\n \n Basic Protocol 3<\/jats:bold>\n : Measurement of mitochondrial membrane potential using the Muse MitoPotential assay\n <\/jats:p>\n \n Basic Protocol 4<\/jats:bold>\n : Ultrastructural evaluation of mitochondrial damage by transmission electron microscopy\n <\/jats:p>\n \n Basic Protocol 5<\/jats:bold>\n : Evaluation of lipid damage using a human 8\u2010iso prostaglandin F2\u03b1 ELISA\n <\/jats:p>\n \n Basic Protocol 6<\/jats:bold>\n : Evaluation of total superoxide dismutase activity using an activity assay kit\n <\/jats:p>\n \n Basic Protocol 7<\/jats:bold>\n : Evaluation of catalase activity using an activity assay kit\n <\/jats:p>\n \n Basic Protocol 8<\/jats:bold>\n : Immunoblot analysis of AKT to assess PI3K\/AKT signaling\n <\/jats:p>","DOI":"10.1002\/cpz1.70328","type":"journal-article","created":{"date-parts":[[2026,3,19]],"date-time":"2026-03-19T11:19:11Z","timestamp":1773919151000},"source":"Crossref","is-referenced-by-count":0,"title":["Analysis of Reactive Oxygen Species\u2013Induced Cellular Damage in Cervical Cancer"],"prefix":"10.1002","volume":"6","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-8858-8633","authenticated-orcid":false,"given":"Hoi Ching Cherry","family":"Chan","sequence":"first","affiliation":[{"name":"Department of Chemical Pathology, Faculty of Health Sciences University of Pretoria Pretoria Gauteng South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7050-4974","authenticated-orcid":false,"given":"Sachin","family":"Bhoora","sequence":"additional","affiliation":[{"name":"Vitamin D Cancer Group, Division of Chemical Pathology, Department of Pathology, Faculty of Health Sciences University of Cape Town Cape Town South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3652-1193","authenticated-orcid":false,"given":"Esther","family":"Zhou","sequence":"additional","affiliation":[{"name":"Vitamin D Cancer Group, Division of Chemical Pathology, Department of Pathology, Faculty of Health Sciences University of Cape Town Cape Town South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3953-227X","authenticated-orcid":false,"given":"Sumari","family":"Marais","sequence":"additional","affiliation":[{"name":"Department of Physiology, Faculty of Health Sciences University of Pretoria Pretoria Gauteng South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1570-3959","authenticated-orcid":false,"given":"Rivak","family":"Punchoo","sequence":"additional","affiliation":[{"name":"Department of Chemical Pathology, Faculty of Health Sciences University of Pretoria Pretoria Gauteng South Africa"},{"name":"Vitamin D Cancer Group, Division of Chemical Pathology, Department of Pathology, Faculty of Health Sciences University of Cape Town Cape Town South Africa"},{"name":"Division of Chemical Pathology, Department of Pathology, Faculty of Health Sciences University of Cape Town Cape Town South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,3,19]]},"reference":[{"key":"e_1_2_13_2_1","doi-asserted-by":"publisher","DOI":"10.1098\/rstb.2015.0434"},{"key":"e_1_2_13_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biopha.2024.117084"},{"key":"e_1_2_13_4_1","doi-asserted-by":"publisher","DOI":"10.1089\/ars.2022.0173"},{"key":"e_1_2_13_5_1","doi-asserted-by":"publisher","DOI":"10.7150\/ijbs.49514"},{"key":"e_1_2_13_6_1","doi-asserted-by":"publisher","DOI":"10.1007\/978\u20101\u20100716\u20102376\u20103_3"},{"key":"e_1_2_13_7_1","doi-asserted-by":"publisher","DOI":"10.1186\/s11658\u2010021\u201000299\u20100"},{"key":"e_1_2_13_8_1","doi-asserted-by":"publisher","DOI":"10.3791\/62663"},{"key":"e_1_2_13_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cub.2014.03.034"},{"key":"e_1_2_13_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.freeradbiomed.2016.03.001"},{"key":"e_1_2_13_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbamcr.2022.119317"}],"container-title":["Current Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70328","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/cpz1.70328","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70328","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T02:15:48Z","timestamp":1789438548000},"score":1,"resource":{"primary":{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/10.1002\/cpz1.70328"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3]]},"references-count":10,"journal-issue":{"issue":"3","published-print":{"date-parts":[[2026,3]]}},"alternative-id":["10.1002\/cpz1.70328"],"URL":"https:\/\/doi.org\/10.1002\/cpz1.70328","archive":["Portico"],"relation":{},"ISSN":["2691-1299","2691-1299"],"issn-type":[{"value":"2691-1299","type":"print"},{"value":"2691-1299","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,3]]},"article-number":"e70328"}}