Fig 2: AMPK activation restores Aß42-induced mitochondrial hyperfission and inflammasome activation in LPS-primed microglia. LPS-primed microglia were pretreated with AICAR (1 mM) for 1 h and then treated with Aß42 for 24 h. (a–e) Representative immunoblot (a) and densitometric quantifications (b–e) of the protein levels of pAMPK, AMPK, pSyk, Syk, Drp1, pro-IL-1ß, and ß-actin in total cell lysate. (f–g) MitoSOX (red) and DAPI (blue) staining and quantification of MitoSOX fluorescence intensity. Scale bar: 10 µm. (h) Immunostaining of ASC (red) and DAPI (blue). Scale bar: 10 µm. (i) Percentages of microglia containing ASC foci were counted. Data are shown as mean ± SEM from at least three independent experiments and are analyzed by one-way ANOVA and Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Fig 3: Gene expression analysis for TBI and control mice. Volcano plot of differential analysis (A). Heatmap of differential analysis (B). Comparison of TNF-a, IL-1ß, IL-6, NLRP3, and ASC expression between TBI group and control group (C) (n = 5, Kruskal-Wallis test). Bubble chart of BP, CC, and MF analysis (D). Bubble chart of KEGG analysis (E). Enrichment plots for GO and KEGG items from gene set enrichment analysis (GSEA) (F,G). Heatmap of immune infiltration in sham and TBI groups (H). Violin plot of immune infiltration in sham and TBI groups (I). All data are shown as mean ± SD. * p < 0.05, ** p < 0.01.

Fig 4: NLRP1 activation is mediated by its ubiquitination. Protein expression levels of NLRP1 (a) in HaCaT cells pre-treated or not with the proteasome inhibitor MG-132 and then exposed to O3. (b) Double immunofluorescence staining for NLRP1 (green) and ASC (red), in HaCaT cells pre-treated with MG-132 and then exposed to O3. Blue staining (DAPI) represents nuclei. Images were taken at 40 × magnification (scale bar = 20 µm). The fluorescent levels were quantified using ImageJ software. Protein expression levels of p20 Caspase 1 over Pro-Caspase 1 (c), and UBR2 (d) in HaCaT cells pre-treated with MG-132 and exposed to O3. (e) Double IF staining for NLRP1 (red) and UBR2 (green) in HaCaT cells pre-treated with MG-132 and exposed to O3. Blue staining (DAPI) represents nuclei. Images were taken at 40 × magnification (scale bar = 20 µm). For all the experiments HaCaT cells were pre-treated or not with the proteasome inhibitor MG-132 20 µM for 2 h and then exposed to O3 0.4 ppm for 1 h. Samples were collected at the indicated timepoints post 1-h O3 exposure. For all western blotting the protein expression level was quantified using ImageJ software and ß-actin or Red ponceau were used as internal control. Data are the results of the averages of at least three di?erent experiments, *p < 0.05 and #p < 0.05 by 2-way ANOVA followed by Tukey’s post-hoc comparison test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig 5: Role of inflammasome in glucose fluctuation-induced myocardial fibrosis. (A) Immunofluorescence staining of intracellular NLRP3–ASC interaction examined by confocal microscopy. The NRCFs were labeled with anti-NLRP3 (red), anti-ASC (green), and DAPI (blue) (n = 3 per group). (B–G) After siRNA targeting NLRP3 transfection, the protein expressions of NLRP3, cleaved caspase-1 and collagen I were measured by western blot (n = 3 per group). (H) Representative images of immunofluorescence staining of intracellular a-SMA in the NG, HG, GF, and GF + siRNA groups (n = 3 per group).