AIM2 Antibody - #DF3514

FIGURE 1. The innate immune sensor AIM2 expression level is positively correlated with the prognosis of BLCA patients. (A) Kaplan-Meier curves showing the overall survival (OS) time of patients with BLCA in the TCGA cohort. (B) Kaplan-Meier curves presenting the progression-free survival (PFS) of patients with BLCA in the TCGA cohort. (C) H and E staining of human high- and low-grade BLCA samples. (D) The protein level of AIM2, EpCAM (representing the epithelial component of bladder cancer), and Vimentin (the biomarker of the stromal component) by 4-color multiplex immunofluorescence (MIHC) staining assays. Survival rate (A,B) is compared by Log-Rank (Mantel-Cox) test.

FIGURE 3. AIM2-overexpression in 5637 cells enhanced inflammasome activation. (A) Immunoblotting showing protein level of flag-tagged AIM2 in AIM2-overexpressed 5637 cells and the control. (B) Immunofluorescence images of AIM2-overexpressed 5637 cells and the control after transfected with or without poly (dA:dT) for 3 h. White arrows indicate the ASC specks. (C) Immunoblot analysis of p20 in the supernatants (SN), and pro-caspase-1, pro-IL-1β, and GSDMD in the lysates of poly (dA:dT)- treated 5637 cells. (D) ELISA of IL-1β in the supernatants of 5637 cells after poly (dA:dT) stimulation. (E) ELISA of IL-18 in the supernatants of 5637 cells after poly (dA:dT) stimulation. (F) Lactate Dehydrogenase (LDH) release in the supernatants of poly (dA:dT) stimulated 5637 cells, detected by diaphorase catalyzed iodonitrotetrazolium chloride (INT) color reaction. In (D–F), the cells were treated with poly (dA:dT) (1 μg/ml, 9 h) or not. Data in (D–F) are presented as the means ± SEM. p-values in (D–F) were calculated using the student’s t-test. * indicates p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001; ns, not significant (p > 0.05).

Figure 4 Rg1 inhibits pyroptosis through STAT3 signaling. (A) Conserved sequences at binding sites of STAT3. (B) STAT3 binding sites on AIM2 promoters predicted by JASPAR. (C) The binding relationship between STAT3 and AIM2 promoter verified by ChIP-qPCR experiment. *p < 0.05 compared with IgG negative control. (D) Western blotting analysis showing NLRP3, ASC, AIM2, cleaved-caspase-1, pro-caspase-1, IL-1β, mature-IL-1β, GSDMD and GSDMD-N protein expression in BV-2 cells with or without Rg1 pretreatment exposed to LPS or LPS with IL-6 stimulation, and with or without stattic treatment induced by LPS. (E–M) Bar graph shows gray value analysis based on immunoblot images. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control, #p < 0.05 and ###p < 0.001 compared with LPS treatment group, ∆p < 0.05 and ∆∆∆p < 0.001 compared with LPS+Rg1 treatment group. (N) Flow cytometry shows the percentage of dead cells over total cells in each stage. The effect of Rg1 (60μM) in down-regulating the rate of apoptotic cells induced by LPS was abrogated by IL-6. Compared with LPS (2μg/mL) treatment group, the rate of apoptotic cells was markedly decreased in stattic and LPS co-incubated group. (O) Bar graph shows the statistical results of the rate of dead cells in each group. **p < 0.01 compared with control, ##p < 0.01 compared with LPS treatment group, ∆∆p < 0.01 compared with LPS+Rg1 treatment group.

Fig. 1Elevated expression of pyroptosis-related proteins in the prefrontal cortex of the brain of HIV-1-infected METH abusers. (A) WB analysis of GSDMD and GSDMD-N protein expression in the prefrontal cortex of both groups. (B) The expression of AIM2, IL-1β, IL-18, and Caspase-1 p20 proteins in the prefrontal cortex. (C) IBA1 expression in IHC-stained the prefrontal cortex (scale bars, 50 µm). (D) IBA1 expression in IF-stained the prefrontal cortex (scale bars, 50 µm). The data are presented as mean ± SEM. n ≥ 3 per group (3 different individuals). *: p 

Figure 3 Calpeptin inhibits AIM2 and NLRP3 inflammasome-mediated inflammation, and upregulates Klotho protein expression in IR mouse model. (A) Western blotting of Calpain 1, Calpain 2, Klotho, AIM2, NLRP3, pro-Caspase 1, cleaved-Caspase 1, IL-1β and IL-18 in the kidney of all mice. (B) Quantitative determination of Calpain 1, Calpain 2, Klotho, AIM2, NLRP3, cleaved-Caspase 1 and IL-18. (C) The mRNA levels of Calpain 2, Klotho, AIM2, ASC and GSDMD in the kidney among different groups. (D) The calpain activity of renal issues was measured by the relative fluorescence units (400/505 nm). (E) The cathepsin B activity of kidney issues was tested by the relative fluorescence units (400/505 nm). (F) Representative immunohistochemical micrographs from the kidney issues of different groups stained with Calpain 1, Calpain 2 and AIM2. ×400, bar = 50 μm. All data were presented as mean ± SEM (n = 3). NS, no significance; *p < 0.05 vs. sham group; **p < 0.01 vs. sham group; ***p < 0.001 vs. sham group; #p < 0.05 vs. IR group; ##p < 0.01 vs. IR group; ###p < 0.001 vs. IR group. CP, calpeptin; IR, ischemia/reperfusion; RFU, relative fluorescence units.

Figure 3 Calpeptin inhibits AIM2 and NLRP3 inflammasome-mediated inflammation, and upregulates Klotho protein expression in IR mouse model. (A) Western blotting of Calpain 1, Calpain 2, Klotho, AIM2, NLRP3, pro-Caspase 1, cleaved-Caspase 1, IL-1β and IL-18 in the kidney of all mice. (B) Quantitative determination of Calpain 1, Calpain 2, Klotho, AIM2, NLRP3, cleaved-Caspase 1 and IL-18. (C) The mRNA levels of Calpain 2, Klotho, AIM2, ASC and GSDMD in the kidney among different groups. (D) The calpain activity of renal issues was measured by the relative fluorescence units (400/505 nm). (E) The cathepsin B activity of kidney issues was tested by the relative fluorescence units (400/505 nm). (F) Representative immunohistochemical micrographs from the kidney issues of different groups stained with Calpain 1, Calpain 2 and AIM2. ×400, bar = 50 μm. All data were presented as mean ± SEM (n = 3). NS, no significance; *p < 0.05 vs. sham group; **p < 0.01 vs. sham group; ***p < 0.001 vs. sham group; #p < 0.05 vs. IR group; ##p < 0.01 vs. IR group; ###p < 0.001 vs. IR group. CP, calpeptin; IR, ischemia/reperfusion; RFU, relative fluorescence units.

Fig. 5. Effects of Rg1 treatment on inflammation in LPS-induced CKD mice. (A) Representative WB images of AIM2, Caspase-1 p20, IL-6, and Cleaved-IL-1β. (B) Statistical results of AIM2. (C) Statistical results of Caspase-1 p20. (D) Statistical results of IL-6. (E) Statistical results of Cleaved-IL-1β. (F) Statistical results of Il1b and Casp1 mRNA (qPCR). (G) Representative images of AIM2 (IF, ×400, bar: 20 μm). (H) Statistical results of AIM2 IF. Data are expressed as mean ± SD, WB and IF, n = 4, qPCR, n = 3. **P < 0.01 versus Control; #P < 0.05, ##P < 0.01 versus Model.

Fig. 5. Effects of Rg1 treatment on inflammation in LPS-induced CKD mice. (A) Representative WB images of AIM2, Caspase-1 p20, IL-6, and Cleaved-IL-1β. (B) Statistical results of AIM2. (C) Statistical results of Caspase-1 p20. (D) Statistical results of IL-6. (E) Statistical results of Cleaved-IL-1β. (F) Statistical results of Il1b and Casp1 mRNA (qPCR). (G) Representative images of AIM2 (IF, ×400, bar: 20 μm). (H) Statistical results of AIM2 IF. Data are expressed as mean ± SD, WB and IF, n = 4, qPCR, n = 3. **P < 0.01 versus Control; #P < 0.05, ##P < 0.01 versus Model.