Fig 2: Pyroptosis occurs in VSMCs after exposure to ß-GP.VSMCs were treated with ß-GP (10 mM) for the indicated time. A Calcium deposition in VSMCs was assessed by Alizarin red staining (positive staining: red; scale bar = 100 µm). B Quantitative analysis of calcium deposition in VSMCs normalized to the protein content. C Protein levels of pro-CASP1, cleaved CASP1, GSDMD-N, and IL1B were determined by western blotting. D Quantification of the results shown in C. E The IL1B content in VSMC culture supernatants was determined by ELISA. F The release of LDH was detected using the LDH Assay Kit. G The percentage of PI-positive cells was measured using Hoechst 33342 (blue)/PI (red) double staining (top: Representative images; bottom: Quantitative analysis of PI-positive cells). H CASP1 expression was determined by immunofluorescent staining (top: Representative images; bottom: Mean fluorescence intensity (MFI) of CASP1 was quantified). I The percentage of TUNEL-positive cells (green) was evaluated by TUNEL staining (top: Representative images; bottom: Quantitative analysis of TUNEL-positive cells). Scale bar = 50 µm for (G); Scale bar = 20 µm for (H) and (I). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group; #P < 0.05, ##P < 0.01 vs. indicated group; and N.S. not significant.

Fig 3: Cigarette Smoke (CS) exposure induces GSDMD cleavage in bronchial epithelial cells in an NLRP3-dependent manner. Immunoblot for total and cleaved GSDMD proteins in lung homogenates of Air or CS-exposed mice of WT, Nlrp3-/- or Gsdmd-/- mice (A) and quantification of total GSDMD (B) and cleaved GSDMD (C) immunoblot were shown. Cleaved GSDMD immunostaining was performed on lung sections of Air or CS-exposed WT, Nlrp3-/- and Gsdmd-/- mice (D). Data are representative of three experiments. Bar graph are expressed ± SEM (n =2–4 mice per group).

Fig 4: Both GSDMD-KO and NLRP3-KO reversed the global expression of neuroinflammation- and neuropathology-related genes at 3 days after TBI. (A) PCA analysis of RNA-seq data from WT, GSDMD-KO, and NLRP3-KO mice at 3 days after sham or TBI treatment (n = 3). (B,C) Heatmap analysis of neuroinflammation- and neuropathology-related gene expression patterns from WT, NLRP3-KO, and GSDMD-KO mice at 3 days after TBI.

Fig 5: NLRP3-KO inhibited the expression and cleavage of GSDMD at 3 days after TBI. (A) Representative images of agarose gel electrophoresis for NLRP3-KO mouse genotyping. (B) Representative images of immunoblots for NLRP3 using brain samples from WT mice and NLRP3-KO mice. (C) Representative images of immunoblots for GSDMD, N-GSDMD, and total protein. (D,E) Quantitative analysis of GSDMD and N-GSDMD levels (n = 6). All data are presented as the mean ± SEM and were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test for significance. ***p < 0.001.