Extended Data Figure 1: Ectopic expression of GSDME in HeLa cells switches TNF-induced apoptosis to pyroptosis, and dosage effect of GSDME expression.

a, b, Morphological and flow-cytometry assays of GSDME-induced apoptosis-to-pyroptosis switch. GSDMD^−/− HeLa cells stably expressing the caspase-3-sensitive GSDMD^DEVD or GSDMA3 or GSDMD^−/−CASP3^−/− HeLa cells stably expressing GSDME were assayed and phase-contrast cell images are shown in a. GSDMD^−/− HeLa cells expressing an empty vector or GSDME were stained with propidium iodide and annexin V-Alexa 647 for flow cytometry (b). c, Effects of the caspase-3-specific inhibitor zDEVD on GSDME-mediated pyroptosis in GSDMD^−/− HeLa cells. ATP cell viability and LDH-release cell death are expressed as mean ± s.d. from three technical replicates. d, e, Immunoblotting assays of caspase-3 cleavage of GSDME in GSDMD^−/− or GSDMD^−/−CASP3^−/− HeLa cells. Flag–GSDME or Flag–GSDME1 (wild-type or D256A mutant) were stably expressed in cells. f, g, Two pools of HeLa cells expressing low (GSDME^Low) or high (GSDME^High) levels of GSDME were obtained by flow-cytometry sorting; the immunoblots in f show the expression of GSDME. Cells were labelled by ectopically expressed enhanced green fluorescent protein (eGFP). g, Time-lapse phase-contrast and fluorescent images of cells taken at the indicated time points after stimulation (scale bar, 20 μm). Real-time videos are included in Supplementary Videos 6, 7, 8. All cells were stimulated with TNF + CHX. Data shown are representative of at least three independent experiments.