Journal: Frontiers in Pharmacology

Article Title: Identification of WT161 as a Potent Agent for the Treatment of Colitis by Targeting the Nucleotide-Binding Domain-Like Receptor Family Pyrin Domain Containing 3 Inflammasome

doi: 10.3389/fphar.2022.780179

Figure Lengend Snippet: WT161 decreased the levels of the pro-inflammatory factors in the colonic tissue of DSS-induced colitis mice and activated peritoneal macrophages induced by LPS + DSS. (A,B) The colonic explants isolated from mice in the animal experiments were cultured for 24 h, and the interleukin (IL)-1β (A) and IL-6 (B) levels in the supernatant was detected by ELISA. For comparison, we extracted the same weight of colon explants (DMSO + H 2 O group, n = 3; other groups, n = 7). (C,D) The IL-1β (C) and IL-6 (D) levels in the supernatant culture of LPS + DSS-activated peritoneal macrophages. For (A–D) , the p -values were calculated using one-way ANOVA with the Bonferroni’s test. ** p < 0.01; **** p < 0.0001. DSS, dextran sulfate sodium; DMSO, dimethyl sulfoxide.

Article Snippet: WT161 (cat. no. 1206731-57-8) from Selleck Chemicals; DSS (cat. no. 0216011080, 36,000–50,000 molecular weight) from MP Biomedicals; ultrapure lipopolysaccharide (LPS) ( E. coli 0111: B4, cat. no. tlrl-3pelps), ATP (cat. no. tlrl-atpl), and nigericin (cat. no. tlrl-nig), monosodium urate (MSU) (cat. no. tlrl-msu) from InvivoGen; Protein A/G PLUS-Agarose (cat. no. sc-2003) from Santa Cruz; mouse immunoglobin IgG protein (cat. no. ab198772) from Abcam, and cell lysis buffer (CLB) (cat. no. 9803) from Cell Signaling Technology; mouse IL-1β (cat. no. 88–7013), IL-6 (cat. no. 88-7064-88) ELISA kits (Thermo Fisher).

Techniques: Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Identification of WT161 as a Potent Agent for the Treatment of Colitis by Targeting the Nucleotide-Binding Domain-Like Receptor Family Pyrin Domain Containing 3 Inflammasome

doi: 10.3389/fphar.2022.780179

Figure Lengend Snippet: WT161 decreased NLRP3 inflammasome activation in the peritoneal macrophages. (A) Mouse peritoneal macrophages were pre-treated with WT161, and then primed with lipopolysaccharide (LPS), followed by stimulation with nigericin. The interleukin (IL)-1β in the supernatant was detected by ELISA. (B,C) The level of cleaved caspase-1 (p10) and mature IL-1β (p17) upon nigericin stimulation in the supernatant (SN) by WT161 treatment was analyzed by western blot (B) and quantitative analysis (C) . (D) Immunoblot analysis of ASC oligomerization in lysates of LPS-primed peritoneal macrophages treated with WT161 and then stimulated with nigericin. (E) Quantitative analysis of ASC oligomerization. (F) Immunofluorescence microscopy analysis of ASC Speck (arrow) in LPS-primed peritoneal macrophages treated with or without WT161, and then stimulated with ATP or nigericin. Scale bars: 10 μm. (G) Percentage of cells with ASC specks. The image and data shown in (A–F) are representative of three independent experiments. For (A,C,E,G) , the p -values were calculated using two-way ANOVA with the Bonferroni’s multiple comparisons test. * p < 0.05; **** p < 0.0001; while p > 0.05 displayed as ns. DMSO, dimethyl sulfoxide; LN, LPS + nigericin; SN, supernatant; MOCK, blank control.

Article Snippet: WT161 (cat. no. 1206731-57-8) from Selleck Chemicals; DSS (cat. no. 0216011080, 36,000–50,000 molecular weight) from MP Biomedicals; ultrapure lipopolysaccharide (LPS) ( E. coli 0111: B4, cat. no. tlrl-3pelps), ATP (cat. no. tlrl-atpl), and nigericin (cat. no. tlrl-nig), monosodium urate (MSU) (cat. no. tlrl-msu) from InvivoGen; Protein A/G PLUS-Agarose (cat. no. sc-2003) from Santa Cruz; mouse immunoglobin IgG protein (cat. no. ab198772) from Abcam, and cell lysis buffer (CLB) (cat. no. 9803) from Cell Signaling Technology; mouse IL-1β (cat. no. 88–7013), IL-6 (cat. no. 88-7064-88) ELISA kits (Thermo Fisher).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence, Microscopy