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. 2021 Mar 4;11(9):4531–4548. doi: 10.7150/thno.54803

Figure 5.

Figure 5

PPARγ agonists limit the KDM5-specific H3K4me3 modifications in il-17 gene locus through regulating the GLS1/2-HG axis. (A-B) The naïve CD4+ T cells were prepared and treated with anti-CD3/CD28 in the presence or absence of Th17-skewing cytokines, 2-HG (0.5 mM), rosiglitazone (ROSI; 30 µM) as well as pioglitazone (PIO; 30 µM) for 72 h. The relative mRNA expression levels of RORγt (A) and IL-17A (B) were determined by Q-PCR assay. (C-D) The naïve CD4+ T cells were transfected with GLS1 plasmid, and then treated with anti-CD3/CD28, Th17-skewing cytokines, ROSI (30 µM) or PIO (30 µM) for 72 h. The relative mRNA expression levels of RORγt (C) and IL-17A (D) were determined by Q-PCR assay. (E-F) The naïve CD4+ T cells were treated with anti-CD3/CD28 in the presence or absence of Th17-skewing cytokines, ML324 (0.2 µM), CPI-455 (10 µM), GSK-J4 (20 nM), ROSI (30 µM) as well as PIO (30 µM) for 72 h. The relative mRNA expression of IL-17A was detected by Q-PCR. (G-I) The naïve CD4+ T cells were prepared, and treated with anti-CD3/CD28 in the presence or absence of Th17-skewing cytokines, ROSI (3, 10, 30 µM) as well as PIO (3, 10, 30 µM) for 72 h. The protein level of H3K4me3 was analyzed by western blotting assay (G), and the enrichment of H3K4me3 in promoter (H) and CNS1, 2, 3, 4 (I) region of il-17 gene was analyzed by ChIP. (J-K) The naïve CD4+ T cells were treated with anti-CD3/CD28 in the presence or absence of Th17-skewing cytokines, 2-HG (0.5 mM), ROSI (30 µM) as well as PIO (30 µM) for 72 h. The enrichment of H3K4me3 in promoter (J) and CNS2 (K) region of il-17 gene was analyzed by ChIP. (L-M) The naïve CD4+ T cells were transfected with GLS1 plasmid, and then treated with anti-CD3/CD28, Th17-skewing cytokines, ROSI (30 µM) or PIO (30 µM) for 72 h. The enrichment of H3K4me3 in promoter (L) and CNS2 (M) region of il-17 gene was analyzed by ChIP. (N) The naïve CD4+ T cells were treated with anti-CD3/CD28 in the presence or absence of Th17-skewing cytokines, CPI-455 (10 µM), ROSI (30 µM) as well as PIO (30 µM) for 72 h. The frequency of CD4+IL-17A+ T cells was determined by flow cytometry. Data were presented as the means ± S.E.M. of three independent experiments. #P < 0.05, ##P < 0.01 vs. Control group; *P < 0.05, **P < 0.01 vs. Th17 group (Model group); $P < 0.05, $$P < 0.01 vs. ROSI group; &P < 0.05, &&P < 0.01 vs. PIO group.