Fig 2: Four cysteine residues are required for human GSDMD to sense oxidative stress. (A) Model graph of four oxidized cysteine residues on GSDMD. (B) Cys38, Cys56, Cys268, and Cys467 were oxidized after incubation with H2O2. (C) Immunoblot analysis of the cleavage of WT, 3CS, and 4CS GSDMD mutants by artificially activated CASP1 treated with H2O2 or left untreated. (D) The LDH release assay was used to quantify cell death in 293T-acCASP1 cells overexpressing WT, 3CS, and 4CS GSDMD mutants treated with 100 µM H2O2 for 6 h or left untreated. (E and F) Immunoblot analysis of single CS and 2CS mutants of GSDMD cleavage by artificially activated CASP1 treated with H2O2 or left untreated. (G) Cell culture supernatants were collected for the LDH release assay of single CS and 2CS groups. Data from western blots and LDH assays represent the average of three measurements. The Arabic numerals listed below the western blot images representing the greyscale analysis of GSDMD-N domain with loading control (a-tubulin). Mean ± SD of three experiments is shown. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: Qu inhibits the LPS-induced NLRP3 inflammasome and related pyroptosis in BV2 cells. Qu (30 and 60 µM, 1 h)-pretreated BV2 cells were stimulated with LPS (100 ng/ml, 24 h) and ATP (5 mM, 30 min). (a–c) Expression of NLRP3, pro-caspase-1, pro-IL-1ß, full-length GSDMD and cleaved GSDMD N-terminal in cell lysate (Ly) was detected by an immunoblot assay (a). The relative expression levels were quantified (c). Actin was used as an internal loading control. The cleaved caspase-1 and cleaved IL-1ß in the supernatant (SN) were also detected by an immunoblot assay (a) and quantified as normalized to the control group (b). (d) ELISA of IL-1ß in supernatants from different-treated BV2 cells. (e–f) Treated BV2 cells were costained with YO-PRO-1 (green), a small (629.3 Da) membrane impermeable but pyroptosis-pole permeable dye, and Eth-D2 (red), a larger (1292.7 Da) membrane and pyroptosis-pole impermeable dye, to identify cells with discrete membrane pores. Hoechst staining (blue) indicates total cells. Treatment with 0.1% Triton was used as the positive control. Scale bar, 40 µm. (f) Quantitative analysis of cells that stained positive for YO-PRO-1 and negative for Eth-D2. A total of 15 fields/group (3 wells/group and 5 fields/well) cells were counted. Data are shown as the mean ± SEM of three to four independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 by one-way ANOVA followed by Tukey's post hoc test. Ctl, untreated control; Qu, quercetin; LPS, lipopolysaccharide; Ly, lysate; SN, supernatant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig 4: Gsdmd is not required for the mitochondrial dysfunction in pyroptosis. (A) Immunostaining of the subcellular location of Gsdmd and the mitochondrial protein COX IV in fixed BMDMs in response to inducers of the Nlrp3 inflammasome. Scale bar, 5 µm. (B–D) Primed WT and Gsdmd-deficient BMDMs were treated with 10 µM nigericin for 30 min and then labeled with MitoTracker Deep Red and MitoTracker Green before flow cytometric analysis. (E) Detection of the subcellular location of ROS by labeling with the probe DCFH-DA and MitoTracker CMXRos in live Gsdmd-deficient BMDMs. Scale bar, 5 µm. Data represent the average of three measurements. Mean ± SD of three experiments is shown. NS, P > 0.05, not significant.

Fig 5: E2-induced IL-1ß secretion and pyroptosis. (a) IL-1ß ELISA of MCF-10A cells examined for the concentrations of secreted IL-1ß in the cell culture media following treating the cells with 0–128 nM E2 for 60 h. Four independent experiments were performed. Bars represent +/-SD. (b) IL-1ß ELISA of MCF-10A cells showing the concentrations of secreted IL-1ß in the supernatant following treatment with 32 nM E2 for 1–72 h. Three independent experiments were performed. Bars represent +/-SD. (c) Representative immunofluorescence images of MCF-10A cells following treatment with fluorescently labeled E2 (green) and MitoTracker Red CMXRos (red) to examine the localization of E2 in mitochondria. E2 localized to mitochondria with or without GPER. Blue staining, Hoechst. Scale bars = 5 µm. (d) Cell-based ROS assay to measure ROS in MCF-10A cells following treatment with 32 nM E2 for 15 or 30 min. Antimycin A, an inhibitor of complex 3 of the mitochondrial electron transport chain, was included as a positive control for ROS production, and N-acetyl cysteine was included as an antioxidant control. Four independent experiments were performed. Bars represent +/-SD. DATA were analyzed using a Mann-Whitney U test. *p values less than 0.05 were considered statistically significant. (e) Caspase-1 inflammasome assay was used to measure caspase-1 activity in MCF-10A cells after adding 32 nM E2 for 24 h. YVAD-CHO was used as a caspase-1 inhibitor. Three independent experiments were performed. Bars represent +/-SD. DATA were analyzed using a Mann-Whitney U test. *p values less than 0.05 were considered statistically significant. (f) Western blotting of MCF-10A cells transfected with p3xFLAG-GSDMD to investigate full-length FLAG-GSDMD and cleaved FLAG-GSDMD (31 kDa) using the FLAG antibody following treatment with 32 nM E2 for 0–4 h. (g) Western blotting of MCF-10A cells showing endogenous WT-GSDMD (full length) and cleaved GSDMD following treatment with 32 nM E2 for 0–4 h using an antibody that recognizes the GSDMD-N-terminal. (h) Confocal images of MCF-10A cells treated with 32 nM E2 for 48 h (bottom) or without 32 nM E2 (up) and stained with the GSDMD antibody (green) to investigate GSDMD (N-terminal) distribution on the plasma membrane. Two confocal cellular cross sections are shown. Scale bars = 10 µm. (i) SEM electron microscopy imaging of MCF-10A cells treated with 32 nM E2 for 72 h showing pyroptotic bodies on the surface of the plasma membrane. Scale bars = 1 µm. The presented blots were cropped. Full-length blots are presented in Supplementary Fig. 7.