Fig. 2: GSDMD-NT is sufficient for calcium-induced pyroptotic filopodia, which sprout independently of cell rupture.

a,b, Quantification of filopodia per cell from macrophages in which pyroptosis was stimulated (LPS + NIG, a; PAM + LPS, b). Data were analyzed using a Kruskal–Wallis test with Dunn’s multiple testing correction (n = 75 cells per condition from three independent biological experiments). c, Quantification of Fluo-4 (Ca²⁺ reporter) intensity extracted from a 45 min acquisition following nigericin-induced pyroptosis in BMDM, relative to the first frame in which the cells become positive for SiR-actin (time 0 min). A total of 72 cells per condition were analyzed pooled from three independent biological experiments. Data are mean intensity (in arbitrary units, a.u.) ± s.e.m. d, Spinning disk confocal imaging of macrophages incubated with Fluo-4 and SiR-actin. WT or Gsdmd^−/− LPS-primed BMDM were left untreated or exposed to nigericin. Yellow arrows indicate cells with a spike in calcium that precedes cell rupture. Images are representative of n = 3 independent biological experiments. e, Quantification of filopodia from macrophages pretreated with 2 mM EGTA 1 h before nigericin in Extended Data Fig. 5c. Data were analyzed using a Kruskal–Wallis test with Dunn’s multiple testing correction (n = 75 cells per condition from three independent biological experiments). f, Live fast Airyscan confocal imaging of WT versus Ninj1^−/− LPS-primed BMDM expressing the PLCδ-PH-GFP probe, labeled with SiR-actin and stimulated with nigericin for up to 30 min. Images are Z-stack MIPs and representative of n = 3 independent biological experiments, analyzed by an unpaired two-sided Mann–Whitney test. g, Quantification of filopodia from nigericin-treated BMDM. Tile scan images were collected between 20–30 min post-NLRP3 activation from n = 3 independent biological experiments. Filopodia were counted in SiR-actin-positive cells. h,i, LPS-primed iBMDM stably expressing Tet-GSDMD-NT were exposed to doxycycline ± EGTA-AM, and analyzed for cell lysis, presented as mean of technical replicates (h) or filopodia (i). Filopodia were quantified from 40 randomly chosen cells per condition and time point, corresponding to Extended Data Fig. 5d, and analyzed by a Mann–Whitney two-sided test with a false discovery rate multiple testing correction. Scale bars, 10 µm. Violin plots show mean (solid line) and first and third quartiles (dotted lines). Statistical significance ****P ≤ 0.0001.