Extended Data Fig. 7: GSDMD KO iBMDMs reconstituted with uncleavable D275A GSDMD-FL induce ROS, pyroptosis, and membrane localization upon inflammasome activation and/or ROS activators.
From: ROS-dependent S-palmitoylation activates cleaved and intact gasdermin D
a, Palmitoylation in iBMDMs treated or not with ROS activators or quencher. b,c, ROS activators alone induced significant PI positivity as measured by Incucyte® (b) and increased LDH release (c) in GSDMD KO iBMDMs stably reconstituted or not with GSDMD-GFP D275A or WT GSDMD. d-f, Palmitoylation (d), PI positivity (e), and MitoSOX (f) in GSDMD KO iBMDMs reconstituted with GSDMD-GFP D275A, clone 5, activated by LPS and nigericin, and treated or not with additional ROS activators, quenchers, or 2-BP. g,h, GFP imaging (g) and quantification (h) for GSDMD membrane localization in clone 5 of GSDMD KO iBMDMs reconstituted with GSDMD-GFP D275A upon inflammasome activation and with or not ROS modulators and 2-BP. Scale bars represent 5 μm (g). All results were obtained from at least 3 independent experiments. GSDMD KO iBMDM clones reconstituted with D275A that had equal expression levels to endogenous GSDMD in WT iBMDMs were selected. Error bars represent SEM. Statistics used two-tailed Student’s t-tests, with NS (non-significant) for p > 0.05, * for p < 0.05, ** for p < 0.01, *** for p < 0.001, and **** for p < 0.0001. Immunoblots were incubated with 1:1000 anti-GSDMD antibody and 1:5000 anti-GAPDH antibody for GAPDH loading controls.
