Fig. 3: Methylcobalamin specifically inhibits GSDME-mediated pyroptosis.

a, c, d, e HepG2 were pretreated with indicated concentrations of each compound for 2 h before adding 200 μM deoxycholic acid (DCA) for 4 h, the cytotoxicity of HepG2 cells was determined by LDH release assay (a). Pyroptosis was measured by SYTOX green uptake in the presence of 20 μM MeCbl or DMF (c). Representative immunoblotting analysis of caspase-3 and GSDME in HepG2 (d), and caspase-3 activity was assessed with substrate Ac-DEVD-pNA (e). b Bone marrow derived macrophages (BMDMs) were pretreated with indicated concentrations of MeCbl for 2 h before challenged with LPS transfection by Fugene HD for GSDMD-mediated pyroptosis. Cytotoxicity was determined by LDH release assay. f Changes in HepG2 cell morphology were observed with a microscope (scale bar= 10 μm). g Representative images of GSDME localization in HepG2 cells treated as indicated by confocal microscopy (scale bar= 5 μm). h, i Mice primary hepatocytes were pretreated with indicated concentrations of each compound for 2 h before challenged with 40 nM perforin (PFR) and 0.5 μM GzmB simultaneously for 24 h to activate GSDME-mediated pyroptosis, the cytotoxicity of hepatocytes was determined by LDH release assay (h). Pyroptosis was measured by SYTOX green uptake in the presence of 20 μM MeCbl or DMF (i). j Effect of MeCbl on PFR+GzmB-induced LDH release in hepatocytes pretreated with Z-DEVD-FMK (20 μM). k, m WT, Gsdmd^-/-, Gsdme^-/- hepatocytes were pretreated with indicated inhibitors for 2 h before challenged with Fugene HD/LPS for 16 h (k) or 200 μM DCA for 4 h (m), cytotoxicity was determined by LDH release assay. l, n Representative immunoblotting analysis of GSDMD and GSDME in hepatocytes. Data are mean ± SEM (n = 3) of at least two independent experiments for bar and line charts. Blots and micrographs are representative of three independent experiments. Analysis was done using one-way ANOVA (e) or two-way ANOVA (a–c, h–k, m). p < 0.05 is considered significant to the indicated group and p values are provided in graphs, n.s = nonsignificant compared to vehicle (e) or indicated group (k, m). Source data are provided as Source Data file.