Fig 1: The absence of AIM2 results in increased activation of JNK and ERK pathways.A, B WT and AIM2−/− mice aged 30–32 weeks were administered either PBS or APAP for 1, 2, 3 h, and C, D for 24 h. E, F WT and AIM2−/− mice aged 6–8 weeks were supplemented with PBS or APAP for 24 h. Protein expression was analyzed using western blotting, and phosphorylated proteins were normalized to total proteins. A representative Western blot from three independent experiments is shown. Immunoblot analysis of PCNA in liver homogenates was conducted (G), and quantification of Evans blue dye extravasation was performed at 24 h after APAP injection (H). The number of mice in each group ranged from 9 to 10, and the statistical significance levels are indicated as follows: *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 2: The absence of AIM2 leads to changes in APAP metabolism and enhances inflammation.A CYP2E1 protein expression and B Immunohistochemical staining of NAPQI-protein adducts were performed, and the images were captured at a 100 µm scale bar. C The mtDNA/nDNA content and D the expression of ND1, TFAM, and CYTB were measured in the liver tissues after 3 h of PBS or APAP supplementation. E Total liver GSH levels were also determined. F Cytoplasmic and mitochondrial proteins from 293 T cells were isolated, and AIM2 was analyzed through western blotting. β-tubulin and VDAC1 served as markers for the cytoplasm (Cy) and mitochondria (Mi), respectively. “Wh” designates the whole lysate. G Co-localization analysis of AIM2 and mitochondria in 293 T cells. AIM2 is highlighted in green, mitochondria in red, DAPI in blue, and co-localized regions are depicted in yellow. Scale bars, 10 µm. H TUNEL staining was conducted 24 h after APAP injection, and the images were captured at a 100 µm scale bar. I The serum IL-6 level and J the relative hepatic mRNA levels were measured in WT and AIM2−/− mice at 24 h after APAP supplementation. Each group consisted of six mice per time point. Statistical analysis indicated no significance (Ns), *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 3: RNA-seq of WT and AIM2−/− mice aged 6–8 and 30–32 weeks after APAP treatment.The analysis of RNA-seq data resulted in the Venn diagrams of A the total number and B the differentially expressed genes (DEGs) in four groups. The statistically significant DEGs were plotted in the four comparisons (C) and visualized in the heatmap (D), with relative mRNA expression per row (red, high; blue, low). The fold alteration in gene expression (log2FC) and adjusted P value (−log10) were plotted in the scatter plot between the livers of WT-APAP (30 w) and AIM2−/−-APAP (30 w) group (E) and AIM2−/−-APAP (8w) and AIM2−/−-APAP (30 w) group (F). The KEGG pathways of DEGs in WT-APAP (30 w) and AIM2−/−-APAP (30 w) group (G) and AIM2−/−-APAP (8w) and AIM2−/−-APAP (30 w) group (H) were ranked by P value. The GO functional enrichment of upregulated DEGs in the livers of APAP-treated AIM2−/− mice was conducted (I), with a false discovery rate (FDR) of <0.05 and ±2 fold change (FC).
Fig 4: AIM2 deficiency promotes ERK signaling activation in macrophages and inhibits autophagy pathway activation in hepatocytes.A Western blot analysis of cleaved caspase-3 and 8 in primary hepatocytes from WT and AIM2−/− mice aged 30 weeks stimulated with APAP (10 mM). B Primary hepatocytes from 30-week-old mice were treated with 10 mM APAP for 6 and 24 h, and culture supernatants were collected for AST and ALT measurement. C, D Bone marrow-derived macrophages (BMDMs) from 30-week-old mice were treated with HMGB1 (200 ng/mL), and cell proteins were collected for Western blotting. E–H TAMH cells were transfected with poly(dA:dT) or Flag-AIM2 for 24 h and then stimulated with 10 mM APAP. Cell lysates were obtained for Western blot analysis. Data represent the mean ± SEM of three independent experiments. **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig 5: AIM2 deficiency influences the infiltration of inflammatory cells upon liver injury.A Fluorescence-activated cell sorting (FACS) strategy of immune cells. B, C Liver non-parenchymal cells (LNPCs) separated from WT and AIM2−/− mice aged 30 weeks treated with APAP for 24 and 72 h for flow cytometry analysis. IMs were labeled as CD11b+ ly6chigh and CD11b+ ly6clow. Neutrophils and macrophages were labeled as CD11b+Ly6G+ and CD11b+F4/80+, respectively. Data represent the mean ± SEM of three independent experiments. N = 8–9 mice per group. *P < 0.05; ***P < 0.001.
Supplier Page from Sino Biological, Inc. for Mouse AIM2 Gene ORF cDNA clone expression plasmid, N-Flag tag