Fig. 1: HGS-ETR1/2 induces pyroptosis mediated by cleavage of GSDME.

a After HLCZ01 cells were treated with different concentrations of HGS-ETR1 (top) and HGS-ETR2 (bottom) for 8 h, PI/Hoechst stain was added to the cell supernatant, and the cell death was detected by fluorescence microscopy. The PI-positive cells (dead cells) showed red fluorescence. Scale bar, 125 μm. b, c After HLCZ01 cells were treated with different concentrations of HGS-ETR1 (b) and HGS-ETR2 (c) for 8 h, the cell medium was collected and centrifuged, and the supernatant was collected to detect LDH release. %LDH release = sample A490 nm/ positive control A490 nm*100. d, e After adding different concentrations of HGS-ETR1 (d) and HGS-ETR2 (e) to HLCZ01 cells for 8 h, they were photographed by optical microscopy. The red arrows indicate the morphology of the cells when pyroptosis occurred. Scale bar, 25 μm. f, g After adding different concentrations of HGS-ETR1 (f) and HGS-ETR2 (g) to the cells for 8 h, the dead cells suspended in the medium were collected by centrifugation, and the total protein was obtained by lysis together with the adherent cells. The expression levels of PARP, GSDMD, GSDME, caspase-3 and cleaved caspase-3 were detected by western blot, and actin was used as the internal reference protein. Three independent biological replicates were performed for each of the above experiments. One-way ANOVA was used to analyse significant differences (b, c) (**p <0.01, ***p <0.001, ****p <0.0001).