Fig 1: PEDF inhibits the activation of the NLRP3 inflammasome and reduces downstream increases in IL-18 and IL-1β through downregulation of CMPK2 in the HTR8/SVneo human chorionic trophoblast cell line. (A) The NLRP3 inflammasome in HTR8/SVneo cells was immunoprecipitated using an NLRP3 antibody, and the cell lysate input controls were tested by western blotting. The HTR8/SVneo cells were treated with 10 μg/ml LPS for 6 h. Densitometry analysis of (B) PEDF and CMPK2, (C) NLRP3, ASC and Casp1, and (D) pro-IL-18 and pro-IL-1β protein expression in HTR8/SVneo cells; n=5. (E) Relative mRNA levels of pro-IL-18 and pre-IL-1β in HTR8/SVneo cells; n=5. (F) The protein concentration of IL-18 and IL-1β in the cultured supernatants was analyzed by ELISA. n=5. **P<0.01 vs. relative normal group; #P<0.05, ##P<0.01 vs. relative LPS group; &P<0.05, &&P<0.01 vs. relative LPS + PEDF group. PEDF, pigment epithelium-derived factor; NLRP3, nucleotide-binding oligomerization domain-like receptor protein 3; LPS, lipopolysaccharide; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; CMPK2, cytidine monophosphate kinase 2; Casp1, caspase 1; CMPK2 group, LPS + PEDF-LVs + CMPK2-LV; LV, lentivirus; IP, immunoprecipitation; IB, immunoblotting.
Fig 2: Effects of THP on ER stress and inflammasome activation in golden hamsters with HLP induced by a high-fat diet. (a) The mRNA levels of caspase 12, GRP78, and CHOP in liver tissues were evaluated by qRT-PCR. (b) The protein levels of caspase 12, GRP78, and CHOP in liver tissues were measured by western blotting, with β-actin as a loading control. (c) Serum IL-18 was determined by ELISA, and the results are expressed as the mean ± SD (n = 8). (d) The protein levels of NLRP3, ASC, pro-IL-1β, and IL-1β in liver tissues were measured by western blotting, with β-actin as a loading control. The results of at least three independent experiments are shown as the mean ± SD. M versus NC, ∗∗∗P < 0.001; LD, MD, HD, and PC versus (M) #P < 0.05, ##P < 0.01, and ###P < 0.001.
Fig 3: Melatonin treatment inhibits NLRP3 inflammasome activation. (A) Representative Western blot bands of NLRP3, ASC, and cleaved caspase-1. (B–D) The relative band densities of NLRP3, ASC, and cleaved caspase-1; n = 6 per group. The cropped bands had been run under the same experimental conditions. *P < 0.05 versus sham group, # P < 0.05 versus SAH + vehicle group, and & P < 0.05 versus SAH + Mel group.
Fig 4: The effects of RLX on Lipofectamine-induced CTL siRNA transfection in NLRP3 inflammasome activated BJ3 HDMFs after 72 h. (A) Representative Western blots of NLRP3, ASC, pro-caspase-1, pro-IL-1β, pro-IL-18, α-SMA, and collagen-I protein expression in HDMFs treated with CTL siRNA ± T+L+A ± R treatment after 72 h. (B) Also shown are the mean ± SEM of each end-point measured from each treatment group studied, corrected for GAPDH loading and expressed relative to the value in the CTL siRNA+T+L+A-treated HDMF group, which was expressed as 1 in each case; from n=4–6 separate experiments conducted in duplicate. #p<0.05 ##p<0.01 vs. CTL siRNA+T+L+A-treated group. (C) Additionally shown are the mean ± SEM levels of active caspase-1 (pg/ml) (top panel) and active IL-1β (pg/ml) (bottom panel) in CTL siRNA transfected HDFs treated with T+L+A or T+L+A+R after 72 h.
Fig 5: Free fatty acid-mediated macrophage M1 polarization in vitro. Mouse abdominal macrophage were isolated by high-sugar DMEM and then inoculated into a sterile Petri dish (2.5 × 105/ml). Palmitic acid (PA, 500 μM) was used for fatty acid stimulation. A Inflammation factors were detected by ELISA in macrophage culture medium, which was stimulated by PA, and B quantitative cellular expression was measured. Data are shown as the mean ± SEM, n = 3. *P < 0.05, ANOVA. Protein levels of ASC, IL-1β, IL-18 and NLRP3 were performed by western blot in macrophage stimulated by PA (C). Flow cytometry to assess polarization in macrophage stimulated by PBS (D, E) or PA (F, G). Anti-F4/80 (D, F), anti-CD11c, and anti-CD206 (E, G). CD11c indicates M1 macrophage, CD206 indicates M2 macrophage. Marker genes of macrophage polarization were detected by qRT-PCR (H) and western blotting (I). Data are shown as the mean ± SEM, n = 3. *P < 0.05, ANOVA
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