Extended Data Fig. 1. Inflammasome activation induces NINJ1-dependent PMR downstream of GSDMD.

a, Topology model of non-activated NINJ1 in the plasma membrane (PM)⁸. b, Immunofluorescence microscopy of NINJ1 in BMDMs. Scale bars, 15 µm. c, Western blot analysis of NINJ1 expression in wild-type (WT), Gsdmd^–/– and Ninj1^–/– BMDMs. d, Release of LDH or IL-1β in primed WT, Gsdmd^–/– and Ninj1^–/– BMDMs stimulated with Nigericin (1 h), transfected with poly(dA:dT) (1 h) or LPS (5 h), or infected with S. Typhimurium (1.5 h). Uptake of propidium iodide (PI) was measured every 10 min after the different stimulations. e, Western blot analysis of endogenous GSDMD in primed BMDMs after Nigericin stimulation for 1 h followed by treatment with BS³ crosslinker for 30 min. f, Time-lapse fluorescence microscopy of primed WT and Ninj1^–/– BMDMs upon transfection with LPS. Pyroptotic cells lost membrane integrity (acquisition of PI, a membrane-impermeable DNA dye) and were labeled by annexin-V (labels phosphatidylserine exposure to the outer leaflet of the plasma membrane). Plasma membrane (PM) blebs observed during pyroptosis (white arrows) collapsed in a NINJ1-dependent manner. DIC, differential interference contrast. Scale bars, 20 µm. Graphs show the mean ± SD and data are representative from 2 (b,c,e,f) or pooled from 2 independent experiments performed in triplicate (d). * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. P-values were calculated using one-way ANOVA with multiple comparisons Dunnett tests. For gel source data, see Supplementary Fig. 2.

Source data