Fig 2: Sal and knockdown of NLRP3 attenuates CORT-induced pyroptosis in PC12 cells. (A) NLRP3 was knocked down by NLRP3 siRNA in PC12 cells, transfection efficiency of NLRP3 was detected by western blot (n = 3). (B) The viability of cells was detected by CCK-8 assay (n = 6). (C) Cell cytotoxicity in PC12 cells was detected by LDH cytotoxicity assay (n = 6). (D) Cell viability and cytotoxicity were measured by Calcein-AM/PI staining. Calcein-AM (in green), PI (in red). Original magnification: ×200, Scale bar = 100 µm, n = 3. (E) The levels of cleaved caspase-1, IL-1ß, IL-18, and cleaved GSDMD in PC12 cells were assessed by western blot analysis (n = 3). #### p < 0.0001, ### p < 0.001, ## p < 0.01 compared with the control group; ** p < 0.01, * p < 0.05 compared with the CORT group; ^^^^ p < 0.0001, ^^^ p < 0.001, ^^ p < 0.01, ^ p < 0.05 compared with Si-NC + CORT group.

Fig 3: Cell pyroptosis induced by depletion of PRMT5 was rescued by knockdown of CASP1.A Relative mRNA expression of CASP1 in NCI-H929 cells and U266 cells transfected with the control plasmid (Con) or CASP1 overexpression plasmid (CASP1-OE). B The analysis of Annexin V-FITC and PI by flow cytometry in NCI-H929 (upper panel) and U266 (lower panel) cells transfected with the control or CASP1-OE plasmid. C Western blotting detection of cleaved-CASP1, N-GSDMD, IL-1b, and IL-18 in NCI-H929 and U266 cells transfected with the control or CASP1-OE plasmid. D Relative mRNA expression of CASP1 in NCI-H929 cells and U266 cells transfected with the control plasmid (Con) or CASP1 siRNAs (siCASP1 #1/#2/#3). E The analysis of Annexin V-FITC and PI by flow cytometry in NCI-H929 (upper panel) and U266 (lower panel) cells transfected with PRMT5 shRNA with or without the co-transfected CASP1 siRNAs. F Western blotting detection of Cleaved-CASP1, N-GSDMD, IL-1b, and IL-18 in NCI-H929 and U266 cells transfected with PRMT5 shRNA with or without the co-transfected CASP1 siRNAs. G Weight curves of xenograft mice carrying scr, shPRMT5, or shPRMT5 + siCASP1-transfected NCI-H929 cells. H Images and growth curves of xenograft tumors treated with scr, shPRMT5, or shPRMT5 + siCASP1-transfected NCI-H929 cells. Data shown are mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: ATF4 affects NLRP3 inflammasome-mediated inflammatory response during cerebral I/R injury. (A–E) Representative Western blots showing protein levels of NLRP3 protein, cleaved-caspase-1, cleaved-IL-1ß and cleaved-IL-18. Protein levels were normalized to ß-actin. (F,G) The cytokine levels of IL-1ß and IL-18 were quantified by ELISA assays. (H) The levels of ROS production in the cortex were quantified by a commercial Detection Kit of Reactive Oxygen Species in Tissue. Error bars represent mean ± SD. (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p< 0.0001 vs. indicated group). n = 5 in each group. MCAO, middle cerebral artery occlusion-reperfusion; GFP, green fluorescent protein; AAV, ATF4 overexpression adeno-associated virus; siRNA, small interfering RNA; NC, negative control siRNA.

Fig 5: Estrogen supplementation ameliorates cell pyroptosis by upregulating autophagy via estrogen receptor a. (A) Representative western blots and relative quantitative analysis of Beclin1, LC3B, and SQSTM1 in HVUECs cells that transfected with Control siRNA or ERa siRNA. (B) Representative western blots and relative quantitative analysis of NLRP3, cleaved caspase 1, and GSDMD in HVUECs cells that transfected with Control siRNA or ERa siRNA. (C) Representative western blots and relative quantitative analysis of IL-1ß and IL-18 in HVUECs cells that transfected with Control siRNA or ERa siRNA. (D) Immunofluorescence images of cells that transfected with Control siRNA or ERa siRNA were taken with or without 60 nM Hcy for 12 h and in the presence or absence of 10 nM E2. Green: GSDMD, red: ERa. DAPI was used to stain the nucleus. Mean optical density analysis of GSDMD and ERa was performed. Scale bar: 50 µm. (E) Immunofluorescence images of cells that transfected with Control siRNA or ERa siRNA were taken with or without 60 nM Hcy for 12 h and in the presence or absence of 10 nM E2. Red: Caspase-1. DAPI was used to stain the nucleus. Mean optical density analysis of Caspase-1 was performed. Scale bar: 50 µm. In all experiments, n = 6. **P < 0.01.