Extended Data Fig. 9 |. Macrophages experiments.

a, Comparable protein expression shown by immunoblotting. b, c, Similar sensitivity to pyroptosis and death evasion by glycine protection (b) or low-dose nigericin treatment (c), shown by LDH release (n = 3 biological replicates). d, Cleavage of engineered GSDMA3 chimera (A3chim) by caspase-1 (C1), shown by SDS-PAGE of proteolysis reactions using purified proteins. e, Comparable expression of A3chim (Flag-tagged) and its AP mutants in GSDMD-KO cells. AP1: 4 D/E to A. AP2: 2 D/E to A. f, g, Preferential release of mature IL-1β from glycine-protected living iBMDMs permeabilized by A3chim, shown by immunoblotting (f) and LDH release (n = 3 biological replicates) (g). h, i, IL-1β release from GSDMD-KO iBMDMs expressing with WT or AP-mutant A3chim under glycine protection, shown by immunoblotting (h) and LDH release (n = 3 biological replicates) (i). j-l, IL-1β release from living GSDMD-KO iBMDMs expressing A3chim stimulated by low-dose nigericin, characterized by immunoblotting (j), LDH release (n = 3 biological replicates) (k), and ELISA (n = 3 biological replicates) (l). m, n, IL-1β release from low-dose nigericin-stimulated GSDMD-KO iBMDMs expressing WT or AP-mutant A3chim, evaluated by immunoblotting (m), LDH release (n = 3 biological replicates) (n), and ELISA (n = 3 biological replicates) (o). Data shown in b, c, g, i, k, l, n, and o are mean ± s.d.. Data shown in a, d, e, f, h, j, and m are representative of two independent experiments.