Extended Data Figure 4: Characterization of GSDME deafness mutant (GSDME^deafness) and the switch of chemotherapy drug-induced apoptosis to pyroptosis by caspase-3 cleavage of exogenous GSDME in HeLa cells.

a, b, Pyroptosis-inducing activity of GSDME^deafness. Full-length human GSDME (GSDME-FL), GSDME-N (the caspase-3 cleavage fragment) or GSDME^deafness was transiently expressed in 293T cells. a, Phase-contrast images of the cells. b, ATP cell viability expressed as mean ± s.d. from three technical replicates. The immunoblots in b show the expression of transfected GSDME. c, Half life of 3×Flag-tagged full-length GSDME, GSDME-N and GSDME^deafness in mammalian cells. Twelve hours after transfection of indicated GSDME constructs into 293T cells, 60 μg ml⁻¹ CHX was added to the cells. Total lysates collected at the indicated time points after CHX treatment were subjected to anti-Flag immunoblotting. The triple Flag at the N terminus can inhibit pore-forming activity and thereby inhibit cell death. The upper and lower four panels are from two separate experiments. d, e, Flag-tagged GSDME (wild-type or the D267A or D270A mutant) was stably expressed in GSDMD^−/− HeLa cells or GSDMD^−/−CASP3^−/− HeLa cells. Cells were treated with doxorubucin or 5-FU to induce caspase-3 activation and cell death. d, Phase-contrast images of cell death morphology. e, Anti-Flag and anti-tubulin immunoblotting of total cell lysates. All data shown are representative of at least two independent experiments.