GSDMD Asp₂₇₆ residue is the critical nonredundant cleavage site following caspase-1/11 activation. (A–D) BMDMs were treated with 1 µg/ml Pam3CSK4 for 5 h before stimulation. (A) Immunoblot of GSDMD and actin in combined cell extract (ext) + supernatant (sup) from WT and Gsdmd^D276A/D276A BMDMs after 60 min following control treatment, LPS electroporation, or ATP stimulation for detection of GSDMD full-length (pro), GSDMD NT (p30), and p43. (B) Cell death measured by percent YOYO-1⁺ cells from live cell images taken every 30 min over a 16 h time course following LPS electroporation of BMDMs from two mice per genotype. (C) LDH, IL-1β, and IL-18 release measured from supernatants of BMDMs stimulated with transfected LPS (5 µg/ml plus FuGENE HD), ATP (5 mM), or nigericin (10 µg/ml) after 16 h, 3 h, or 30 min, respectively. (D) LDH, IL-1β, and IL-18 release measured from supernatants of BMDMs infected with indicated strains of bacteria. Data are representative of at least two independent experiments (A and B). Data are presented as mean ± SD (n = 3) and representative of at least three independent experiments (C and D).