Extended Data Fig. 6: GSDMD KO THP-1 cells with uncleavable D275A GSDMD-FL induced ROS, pyroptosis, and membrane localization, and a GSDMD disease mutant enhanced cell death in a palmitoylation-dependent manner.
From: ROS-dependent S-palmitoylation activates cleaved and intact gasdermin D
a,b, ROS activators alone induced significant LDH release (a) and reduced cell viability (b) in GSDMD KO THP-1 stably reconstituted with GSDMD-GFP D275A or WT GSDMD. Expression levels of GSDMD-GFP in reconstituted THP-1 cells were equal to endogenous GSDMD in WT THP-1 cells. c,d, Palmitoylation in THP-1 cells treated or not with ROS activators or quencher (c), or primed and activated and treated or not with ROS activators or quencher (d). e-j, ROS measured by MitoSox (e), palmitoylation (f), LDH release (g), cell viability (h), IL-1β release (i), and cellular ROS (j) in GSDMD KO THP-1 cells or GSDMD KO THP-1 cells reconstituted with D275A, treated or not with ROS activators or quenchers. Expression levels of GSDMD-GFP in reconstituted THP-1 cells were equal to endogenous GSDMD in WT THP-1 cells. All results were obtained from at least 3 independent experiments. Error bars represent SEM. Statistics used two-tailed Student’s t-tests, with NS (non-significant) for p > 0.05, ** for p < 0.01, *** for p < 0.001, and **** for p < 0.0001. Immunoblots were incubated 1:5000 anti-GAPDH antibody for GAPDH, and 1:1000 anti-GSDMD antibody.
