a, GSDME N-terminal (NT) mutations in primary cancer cells in TCGA (https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga). Asterisks denote stop codons; red, truncations; blue, mutants analysed here. b–g, Seventeen GSDME-NT cancer-associated mutations are mapped onto GSDME-NT (amino acids 1–241)—modelled here on the basis of the cryo-electron microscopy structure of the pore conformation of the GSDMA3-NT (Protein DataBank identification code 6CB8)8. These mutations were analysed in two arbitrarily assigned groups (b, e). These 17 mutations and the E245K mutation were analysed, after transient expression of wild-type Flag-tagged GSDME (WT FL GSDME) or wild-type or mutated GSDME-NT (amino acids 1–270) in HEK293T cells, for GSDME expression by immunoblot (c, f) and for LDH release by CytoTox 96 assay (d, g). h–l, Effect of four cancer-related GSDME truncation mutations (resulting in shortened proteins comprising amino acids 1–46, 1–210, 1–451 and 1–491) on GSDME-mediated cell death. GSDME expression of truncated proteins or FL or GSDME-NT (1–270) in HEK293T cells (h, k) and their effect on HEK293T cell death, assessed by morphology using microscopy (i) and by LDH release (j, l). The NT46 truncated protein is too small to be detected in k. Red arrows indicate ballooning pyroptotic cells. m, n, Effect of F2A mutation on GSDME-NT-induced pyroptosis in HEK293T cells. Protein expression is detected by anti-Flag immunoblot (m) and pyroptosis is assessed by LDH release (n). o, p, Effect of transient expression of mouse WT FL GSDME or wild-type or mutated GSDME-NT (mNT270, amino acids 1–270) in HEK293T cells (o) and on LDH release (p). q, r, Effect in 4T1E cells of expression of wild-type or mutated FL mGSDME (q) on raptinal-induced SYTOX green uptake (r). Differences among multiple groups in d, g, j, l, n, p, r were analysed by one-way ANOVA, using the Holm–Sidak method for multiple comparisons. Data are mean ± s.d. of three technical (d, g, j, l, n, p) or biological (r) replicates. P values compare unmutated with mutated constructs. ***P < 0.0001. Data are representative of three independent experiments. Source data