Figure 5: Demethylation Treatment Ameliorated NLRP3 Inflammasome Activation

(A) Immunofluorescence of NLRP3 and α-SMA in ascending aortas of ApoE−/− mice fed a Western diet (0, 12, and 24 weeks). (B) Western blot and quantitative results of NLRP3, pro-IL-1β, and mature IL-1β in aortas of ApoE−/− mice fed a Western diet (0, 6, 12, 18, and 24 weeks). Values are mean ± SEM from 6 animals in each group; *p < 0.05 vs. the 0-week group. (C) Immunofluorescence of NLRP3 and α-SMA in aortas of WT mice fed a 24-week Western diet or ApoE−/− mice fed a 12-week Western diet and treated with 5-aza or phosphate-buffered saline for another 12 weeks (0.25 mg/kg body weight, 3 times per week). Values are mean ± SEM from 6 animals in each group; *p < 0.05. (D and E) Masson and Mac3 immunohistochemistry in aortas of WT mice or ApoE−/− mice treated with 5-aza to analyze plaque components. Values are mean ± SEM from 6 animals in each group; *p < 0.05. (F) BSP and relative methylation rate (20 clones) of the +129 CpG site of the miR-145 promoter in aortas of ApoE−/− mice treated with 5-aza. Values are mean ± SEM from 6 animals in each group; *p < 0.05. (G) Concentration of IL-1β in the serum of ApoE−/− mice treated with 5-aza. Values are mean ± SEM from 6 animals in each group; *p < 0.05. (H) Western blot of NLRP3, pro-IL-1β, and mature IL-1β in vascular smooth muscle cells with demethylation treatment (5-aza, knockdown of DNMT1, or overexpression of Tet2). Values are mean ± SEM of 3 samples in each group; *p < 0.05. α-SMA = α-smooth muscle actin; IL = interleukin; NLRP3 = nucleotide-binding oligomerization domain-like receptor protein 3; WT = wild-type; other abbreviations as in Figures 1, 2, and 3.

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