Fig 2: IRF2 silencing attenuates GSDMD-induced pyroptosis in vivo. (A) Myocardial apoptosis of mice was detected by TUNEL staining. Scale bar, 20 µm. (B) Effect of silencing IRF2 on expression of (B) c-caspase-1, GSDMD-N and GSDMD and (C) IL-1ß and IL-18 in heart tissue of MI model mice. All data are expressed as the mean ± SD (n=3). *P<0.05 vs. Sham; #P<0.05 vs. MI + sh-NC. IRF2, interferon regulatory factor 2; GSDMD, gasdermin D; c-, cleaved; MI, myocardial infarction; sh, short hairpin; NC, negative control.

Fig 3: IRF2 silencing attenuates GSDMD-induced pyroptosis in vitro. (A) IRF-2 expression in H9c2 cells stimulated by hypoxia (6, 12, 24 and 48 h) was detected by reverse transcription-quantitative PCR. *P<0.05 vs. Con. (B) IRF2 expression was detected by western blotting. *P<0.05 vs. sh-NC; #P<0.05 vs. pc-NC. (C) Following transfection of sh-IRF2 and IRF2, apoptosis of H9c2 cells was detected by flow cytometry under hypoxic conditions. &P<0.05 vs. Hypoxia + pc-NC, *P<0.05 vs. Con; #P<0.05 vs. Hypoxia+sh-NC. Following transfection of sh-IRF2 and IRF2, the expression of (D) c-caspase-1, GSDMD-N, GSDMD, (E) IL-1ß, and IL-18 was detected by western blotting under hypoxic conditions. *P<0.05 vs. Con; #P<0.05 vs. Hypoxia + sh-NC. All data are expressed as the mean ± SD (n=3). IRF2, interferon regulatory factor 2; GSDMD, gasdermin D; c-, cleaved; MI, myocardial infarction; sh, short hairpin; NC, negative control; Con, control.

Fig 4: Protein expression of NLRP1, procaspase-1, cleaved caspase-1, and active gasdermin-D (GSDMD-N) in the hippocampus as detected by Western blotting. Full, non-adjusted images of Fig. 8(A,E) see the supplementary material.

Fig 5: NLRP3 was needed in IL6 induced pyroptosis(A) SEM morphology of FE25 cells treated with 100 µM IL-6 for 0, 24, and 36 h.(B) WB assays of NLRP3, cleaved caspase-1, and GSDMD in FE25 were treated with 100 µM IL-6 for 72 h.(C) The expression and intracellular localization of NLRP3 in FE25 cells treated with 100 µM IL-6 with or without MCC950 (NLRP3 inhibitor) and siNLRP3 were detected using confocal microscopy.(D) FE25 cells were stained with Annexin V/propidium iodide (PI) for flow cytometry after treated with 100 µM IL-6 with or without siNLRP3 for 72 h. The expression and intracellular localization of Caspase1 (E), GSDMD (F) and GSDMD-N (G) in FE25 cells treated with 100 µM IL-6 with or without MCC950 (NLRP3 inhibitor) and siNLRP3 were detected using confocal microscopy. Vehicle treated with conditioned medium of NFs treated with 1‰ DMSO.