{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T08:40:59Z","timestamp":1790152859228,"version":"4.0.1"},"reference-count":24,"publisher":"Springer Science and Business Media LLC","issue":"7-8","license":[{"start":{"date-parts":[[2020,6,3]],"date-time":"2020-06-03T00:00:00Z","timestamp":1591142400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2020,6,3]],"date-time":"2020-06-03T00:00:00Z","timestamp":1591142400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Apoptosis"],"published-print":{"date-parts":[[2020,8]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Shikonin induced necroptosis in Jurkat cells were\u00a0identified flow cytometrically by the up-regulation of RIP3 in live cells and that a proportion of these cells underwent other forms of regulated cell death (RCD) which included parthanatos (&lt;\u200910%), or cleaved PARP (&lt;\u200910%) and DNA Damage (&gt;\u200930%). Live necroptotic cells also possessed functioning mitochondria with hyper-polarized mitochondria membrane potential and generated a fivefold increase in cellular reactive oxygen species (ROS) which was resistant to inhibition by zVAD and necrostatin-1 (Nec-1). After loss of plasma membrane integrity these dead necroptotic cells then showed a higher incidence of parthanatos (&gt;\u200940%), or cleaved PARP (&gt;\u200915%) but less DNA Damage (&lt;\u200915%). Inhibition of shikonin induced apoptosis and necroptosis by zVAD and Nec-1 respectively resulted in live necroptotic cells with an increased incidence of cleaved PARP and reduced levels of DNA Damage respectively. Dead necroptotic cells then showed a reduced incidence of parthanatos and DNA Damage after inhibition by zVAD and Nec-1 respectively. A high proportion of these dead necroptotic cells (30%) which lacked plasma membrane integrity also displayed functioning hyper-polarized mitochondria with high levels of cellular ROS and thus had the capacity to influence the outcome of RCD processes rather than just been the end product of cell death, the necrotic cell. Flow cytometry can thus measure multiple forms of RCD and the level of cellular ROS and MMP which highlights the inter-connection between cell death processes and that a single cell may simultaneously display multiple forms of RCD.<\/jats:p>","DOI":"10.1007\/s10495-020-01613-5","type":"journal-article","created":{"date-parts":[[2020,6,3]],"date-time":"2020-06-03T20:02:38Z","timestamp":1591214558000},"page":"548-557","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Flow cytometric detection of hyper-polarized mitochondria in regulated and accidental cell death processes"],"prefix":"10.1007","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7614-6266","authenticated-orcid":false,"given":"G.","family":"Warnes","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2020,6,3]]},"reference":[{"key":"1613_CR1","doi-asserted-by":"publisher","first-page":"14","DOI":"10.1016\/j.semcdb.2014.07.013","volume":"35","author":"W Zhou","year":"2014","unstructured":"Zhou W, Yuan J (2014) Necroptosis in health and diseases. Semin Cell Dev Biol 35:14\u201323","journal-title":"Semin Cell Dev Biol"},{"key":"1613_CR2","doi-asserted-by":"publisher","first-page":"323","DOI":"10.3389\/fphys.2014.00323","volume":"5","author":"KD Marshall","year":"2014","unstructured":"Marshall KD, Baines CP (2014) Necroptosis: is there a role for mitochondria? Front Physiol 5:323","journal-title":"Front Physiol"},{"key":"1613_CR3","doi-asserted-by":"publisher","first-page":"1543","DOI":"10.1038\/aps.2017.112","volume":"38","author":"B Lu","year":"2017","unstructured":"Lu B, Gong X, Wang ZQ, Ding Y, Wang C, Luo TF, Piao MH, Meng FK, Chi GF, Luo YN et al (2017) Shikonin induces glioma cell necroptosis in vitro by ROS overproduction and promoting RIP1\/RIP3 necrosome formation. Acta Pharmacol Sin 38:1543\u20131553","journal-title":"Acta Pharmacol Sin"},{"key":"1613_CR4","doi-asserted-by":"publisher","first-page":"14329","DOI":"10.1038\/ncomms14329","volume":"8","author":"Y Zhang","year":"2017","unstructured":"Zhang Y, Su SS, Zhao S, Yang Z, Zhong CQ, Chen X, Cai Q, Yang ZH, Huang D, Wu R et al (2017) RIP1 autophosphorylation is promoted by mitochondrial ROS and is essential for RIP3 recruitment into necrosome. Nat Commun 8:14329","journal-title":"Nat Commun"},{"key":"1613_CR5","doi-asserted-by":"publisher","first-page":"76","DOI":"10.1111\/imr.12541","volume":"277","author":"B Tummers","year":"2017","unstructured":"Tummers B, Green DR (2017) Caspase-8: regulating life and death. Immunol Rev 277:76\u201389","journal-title":"Immunol Rev"},{"key":"1613_CR6","doi-asserted-by":"publisher","first-page":"1184","DOI":"10.1038\/cdd.2017.65","volume":"24","author":"S Grootjans","year":"2017","unstructured":"Grootjans S, Vanden Berghe T, Vandenabeele P (2017) Initiation and execution mechanisms of necroptosis: an overview. Cell Death Differ 24:1184\u20131195","journal-title":"Cell Death Differ"},{"key":"1613_CR7","doi-asserted-by":"publisher","first-page":"2514","DOI":"10.1101\/gad.13.19.2514","volume":"13","author":"Y Lin","year":"1999","unstructured":"Lin Y, Devin A, Rodriguez Y, Liu ZG (1999) Cleavage of the death domain kinase RIP by Caspase-8 prompts TNF-induced apoptosis. Genes Dev 13:2514\u20132526","journal-title":"Genes Dev"},{"key":"1613_CR8","doi-asserted-by":"publisher","first-page":"77","DOI":"10.1016\/j.canlet.2017.01.004","volume":"390","author":"Z Zhou","year":"2017","unstructured":"Zhou Z, Lu B, Wang C, Wang Z, Luo T, Piao M, Meng F, Chi G, Luo Y, Ge P (2017) RIP1 and RIP3 contribute to shikonin-induced DNA double-strand breaks in glioma cells via increase of intracellular reactive oxygen species. Cancer Lett 390:77\u201390","journal-title":"Cancer Lett"},{"key":"1613_CR9","doi-asserted-by":"publisher","first-page":"107","DOI":"10.1038\/cdd.2011.96","volume":"19","author":"L Galluzzi","year":"2012","unstructured":"Galluzzi L, Vitale I, Abrams JM, Alnemri ES, Baehrecke EH, Blagosklonny MV, Dawson TM, Dawson VL, El-Deiry WS, Fulda S et al (2012) Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012. Cell Death Differen 19:107\u2013120","journal-title":"Cell Death Differen"},{"issue":"24","key":"1613_CR10","first-page":"32","volume":"35","author":"L Galluzzi","year":"2014","unstructured":"Galluzzi L, Keppc O, Krautwaldf S, Kroemerb G, Linkermannf A (2014) Molecular mechanisms of regulated necrosis. Semin Cell Dev Biol 35(24):32","journal-title":"Semin Cell Dev Biol"},{"key":"1613_CR11","doi-asserted-by":"publisher","first-page":"486","DOI":"10.1038\/s41418-017-0012-4","volume":"25","author":"L Galluzzi","year":"2018","unstructured":"Galluzzi L, Vitale I, Aaronson SA, Abrams JM, Adam D, Agostinis P, Alnemri ES, Altucci L, Amelio I, Andrews DW et al (2018) Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Differ 25:486\u2013541","journal-title":"Cell Death Differ"},{"key":"1613_CR12","doi-asserted-by":"publisher","first-page":"133","DOI":"10.1016\/j.molcel.2014.03.003","volume":"54","author":"H Wang","year":"2014","unstructured":"Wang H, Sun L, Su L, Rizo J, Liu L, Wang LF, Wang FS, Wang X (2014) Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3. Mol Cell 54:133\u2013146","journal-title":"Mol Cell"},{"key":"1613_CR13","doi-asserted-by":"publisher","first-page":"1749","DOI":"10.1016\/j.prp.2018.09.002","volume":"214","author":"Z Wang","year":"2018","unstructured":"Wang Z, Guo LM, Wang SC, Chen D, Yan J, Liu FX, Huang JF, Xiong K (2018) Progress in studies of necroptosis and its relationship to disease processes. Pathol Res Pract 214:1749\u20131757","journal-title":"Pathol Res Pract"},{"key":"1613_CR14","doi-asserted-by":"publisher","first-page":"433","DOI":"10.1016\/j.freeradbiomed.2017.04.010","volume":"108","author":"W Sun","year":"2017","unstructured":"Sun W, Wu X, Gao H, Yu J, Zhao W, Lu JJ, Wang J, Du G, Chen X (2017) Cytosolic calcium mediates RIP1\/RIP3 complex-dependent necroptosis through JNK activation and mitochondrial ROS production in human colon cancer cells. Free Radic Biol Med 108:433\u2013444","journal-title":"Free Radic Biol Med"},{"key":"1613_CR15","doi-asserted-by":"publisher","first-page":"132","DOI":"10.1016\/j.bbamcr.2017.10.004","volume":"1865","author":"KH Jang","year":"2018","unstructured":"Jang KH, Jang T, Son E, Choi S, Kim E (2018) Kinase-independent role of nuclear RIPK1 in regulating parthanatos through physical interaction with PARP1 upon oxidative stress. Biochim Biophys Acta Mol Cell Res 1865:132\u2013141","journal-title":"Biochim Biophys Acta Mol Cell Res"},{"key":"1613_CR16","doi-asserted-by":"publisher","first-page":"e23209","DOI":"10.1371\/journal.pone.0023209","volume":"6","author":"Y Cho","year":"2011","unstructured":"Cho Y, McQuade T, Zhang H, Zhang J, Chan FK (2011) RIP1-dependent and independent effects of necrostatin-1 in necrosis and T cell activation. PLoS ONE 6:e23209","journal-title":"PLoS ONE"},{"key":"1613_CR17","doi-asserted-by":"publisher","first-page":"56","DOI":"10.1016\/j.ymeth.2017.10.013","volume":"134\u2013135","author":"HL Lee","year":"2018","unstructured":"Lee HL, Pike R, Chong MHA, Vossenkamper A, Warnes G (2018) Simultaneous flow cytometric immunophenotyping of necroptosis, apoptosis and RIP1-dependent apoptosis. Methods 134\u2013135:56\u201366","journal-title":"Methods"},{"key":"1613_CR18","doi-asserted-by":"publisher","first-page":"453","DOI":"10.1007\/s10495-019-01528-w","volume":"24","author":"D Bergamaschi","year":"2019","unstructured":"Bergamaschi D, Vossenkamper A, Lee WYJ, Wang P, Bochukova E, Warnes G (2019) Simultaneous polychromatic flow cytometric detection of multiple forms of regulated cell death. Apoptosis 24:453\u2013464","journal-title":"Apoptosis"},{"key":"1613_CR19","doi-asserted-by":"publisher","first-page":"1585","DOI":"10.1038\/emboj.2010.43","volume":"29","author":"C Artus","year":"2010","unstructured":"Artus C, Boujrad H, Bouharrour A, Brunelle MN, Hoos S, Yuste VJ, Lenormand P, Rousselle JC, Namane A, England P et al (2010) AIF promotes chromatinolysis and caspase independent programmed necrosis by interacting with histone H2AX. EMBO J 29:1585\u20131599","journal-title":"EMBO J"},{"key":"1613_CR20","doi-asserted-by":"publisher","first-page":"3448","DOI":"10.1016\/j.bbamcr.2013.06.001","volume":"1833","author":"V Nikoletopoulou","year":"2013","unstructured":"Nikoletopoulou V, Markaki M, Palikaras K, Tavernarakis N (2013) Crosstalk between apoptosis, necrosis and autophagy. Biochim Biophys Acta 1833:3448\u20133459","journal-title":"Biochim Biophys Acta"},{"key":"1613_CR21","doi-asserted-by":"publisher","first-page":"101","DOI":"10.15698\/mic2016.03.483","volume":"3","author":"L Galluzzi","year":"2016","unstructured":"Galluzzi L, Kepp O, Kroemer G (2016) Mitochondrial regulation of cell death: a phylogenetically conserved control. Microb Cell 3:101\u2013108","journal-title":"Microb Cell"},{"key":"1613_CR22","doi-asserted-by":"publisher","first-page":"299","DOI":"10.1007\/s10495-018-1455-x","volume":"23","author":"S Chen","year":"2018","unstructured":"Chen S, Lu X, Hu B, Zhao L, Li S, Li Z, Qing X, Liu H, Xu J, Shao Z (2018) Critical contribution of RIPK1 mediated mitochondrial dysfunction and oxidative stress to compression-induced rat nucleus pulposus cells necroptosis and apoptosis. Apoptosis 23:299\u2013313","journal-title":"Apoptosis"},{"key":"1613_CR23","first-page":"85","volume-title":"Flow cytometry select topics","author":"G Warnes","year":"2016","unstructured":"Warnes G (2016) The multiplexing of assays for the measurement of early stages of apoptosis by polychromatic flow cytometry. In: Schmid I (ed) Flow cytometry select topics. On-line: InTech Open, London, pp 85\u201399"},{"key":"1613_CR24","doi-asserted-by":"publisher","first-page":"50","DOI":"10.1016\/j.ab.2017.07.009","volume":"552","author":"L Zorova","year":"2018","unstructured":"Zorova L, Popkov VA, Plotnikov EY, Silachev DN, Pevzner AV, Jankauskas SS, Babenko VA, Zorov SD, Balakireva AV, Juhaszova M, Sollott SJ, Zorov D (2018) Mitochondrial membrane potential. Anal Biochem 552:50\u201359","journal-title":"Anal Biochem"}],"container-title":["Apoptosis"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1007\/s10495-020-01613-5.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/article\/10.1007\/s10495-020-01613-5\/fulltext.html","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1007\/s10495-020-01613-5.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,6,3]],"date-time":"2021-06-03T01:41:37Z","timestamp":1622684497000},"score":1,"resource":{"primary":{"URL":"https:\/\/link.springer.com\/10.1007\/s10495-020-01613-5"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,6,3]]},"references-count":24,"journal-issue":{"issue":"7-8","published-print":{"date-parts":[[2020,8]]}},"alternative-id":["1613"],"URL":"https:\/\/doi.org\/10.1007\/s10495-020-01613-5","relation":{},"ISSN":["1360-8185","1573-675X"],"issn-type":[{"value":"1360-8185","type":"print"},{"value":"1573-675X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,6,3]]},"assertion":[{"value":"3 June 2020","order":1,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Compliance with ethical standards"}},{"value":"The author states he has no conflicting or competing interests.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Conflicts of interest"}}]}}