Fig 2: PCB2 ameliorates nicotine-induced pyroptosis and liver damage in mice. C57BL/6J mice were administrated with PCB2 (50 mg/kg/day) or saline for 6 weeks. After 2 weeks of PCB2 administration, mice were received daily IP injection of nicotine (2 mg/kg/day) or saline for 4 weeks. (A) A schematic illustration of the animal treatment procedure. (B) Serum levels of AST. (C) Serum levels of ALT. (D) Immunoblot analysis of the protein levels of GSDMD, NLRP3 and HMGB1 in liver tissues of the mice. (E) Quantification of GSDMD-NT, NLRP3 and HMGB1 levels as in (D). (F) The activity of Caspase-1 in liver tissues of the mice. (G,H) The concentrations of IL-1ß (G) and IL-18 (H) in the serum of the mice. n = 6 per group; Data shown represent the mean ± SD (*, p < 0.05).

Fig 3: Caspase-1 regulates eCIRP-induced GSDMD-mediated MET formation in THP-1 cells. (A) Caspase-1 is cleaved by the treatment of rmCIRP (1 µg/ml) in THP-1 cells. The cleavage of caspase-1 was observed at 3 and 6 h after the treatment of rmCIRP (1 µg/ml). The Western blot of cleaved caspase-1 was detected first. Full-length GSDMD and ß-actin were probed at the same time after stripping the anti-cleaved GSDMD antibody. The intensity of bands detected was quantified by densitometry. Student t-test: *p <0.05 vs. PBS (without rmCIRP) at 3 h, *p <0.01 vs. PBS (without rmCIRP) at 6 h. (B) The effect of rmCIRP (1 µg/ml) in THP-1 cells is significantly inhibited by z-VAD-fmk (20 µM), a pan-caspase inhibitor. The inhibitor was pre-treated 30 min before rmCIRP (1 µg/ml) treatment, and the cells were subjected to the time-lapse live-cell imaging immediately after the treatment of rmCIRP. The effect of z-VAD-fmk was quantified by counting the cells positive to SYTOX Orange. (C) Treatment of z-VAD-fmk at a dose of 20 µM inhibits the activation of GSDMD by rmCIRP (1 µg/ml). The western blot of cleaved GSDMD was done first. Full length GSDMD and ß-actin were probed at the same time after stripping off the anti-cleaved GSDMD antibody. The band intensity was determined by densitometry and compared to the control group. Full gel images are shown in Supplementary Figure S4 . One-way ANOVA with Turkey method: *p < 0.01 vs. PBS; #p < 0.01 vs. mCIRP for 3 or 6 h treatment study.

Fig 4: Effect of AST IV on the NLRP3/Caspase-1/GSDMD pathway in MCAO/R rats. (a) Western blot assay showing the expression levels of NLRP3, Caspase-1, IL-1ß, GSDMD, and GSDMD-N. (b)–(f) Quantitative analysis of NLRP3, Caspase-1, IL-1ß, GSDMD, and GSDMD-N levels normalized to ß-actin. Values are mean ± SD, n = 5. **P < 0.01 vs. the sham group; #P < 0.05, ##P < 0.01 vs. the MCAO/R group.

Fig 5: Pyroptosis of renal tubular epithelial cells in the CLP-induced S-AKI mouse model. (a) The expression of caspase-1, N-GSDMD, NEK7, and NLRP3 in sham and CLP-induced mice by IHC. (b) The protein expression of NEK7, NLRP3, ASC, active caspase-1, and N-GSDMD by Western blot, and (c) quantification by ImageJ. (d) The expression of inflammatory factors including IL-18, IL-1ß, and THF-a. *P < 0.05, **P < 0.01, and ***P < 0.001.