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. 2019 Apr 11;10:1689. doi: 10.1038/s41467-019-09397-2

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — GSDME augments death receptor-induced pyroptosis and caspase-3 activation. a, b Propidium iodide (PI) uptake (a) and active caspase-3/7 staining (b) in CEM-C7 cells (wild-type (WT), GSDME-knockout (KO), or GSDME-KO reconstituted with WT GSDME-EGFP or GSDME-T6E-EGFP) treated with tumor necrosis factor α (TNFα) plus actinomycin D (ActD) as measured on the IncuCyte over time. c Immunoblots of GSDME, active caspase-3 p17/p19, and high-mobility group box 1 (HMGB1) in combined cell lysates plus media, or HMGB1 released in culture media (media) of WT (WT), GSDME-KO (KO), or GSDME-KO reconstituted with WT GSDME-EGFP (KO+WT) or GSDME-T6E-EGFP (KO+T6E) CEM-C7 cells treated with TNFα plus ActD as indicated. d Immunoblots of cytochrome c (Cyt c) (1st and 2nd panels) released into the culture media (media) or cytosol (Cyto) from WT (WT) or GSDME-KO (KO) CEM-C7 cells treated with TNFα plus ActD for 18 h. e Active caspase-3/7 staining in WT, GSDME-KO, Bid-KO, or GSDME/Bid-dKO CEM-C7 cells treated with TNFα plus ActD as measured on the IncuCyte over time. f Immunoblots of Cyt c (1st and 2nd panels) released into the culture media (media) or cytosol (Cyto) from WT, GSDME-KO (GSE-KO), Bid-KO, or GSDME/Bid-dKO (dKO) CEM-C7 cells treated with TNFα plus ActD. Quantitative analyses of Cyt c release in (d, f) are shown in Supplementary Fig. 17. Results are representative of at least three independent experiments performed in duplicate or triplicate. Error bars represent S.D. Student’s t-test, *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.00005