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. 2021 Jun 1;206(11):2652–2667. doi: 10.4049/jimmunol.2000624

FIGURE 5.

FIGURE 5.

Analysis of histone modifications in NFAT5 target genes in macrophages. (A and B) ChIP analysis of acetylated histone H3 (lysines 9 and 14, AcH3) (A) and acetylated histone H4 (lysines 5, 8, 12, and 16, AcH4) (B) in the promoters of Nos2, Il6, and Tnf in Nfat5+/+ (wild-type) and Nfat5–/– (NFAT5-deficient) BMDM untreated (t = 0) or stimulated with LPS [0.3 ng/ml in (A) and 1 ng/ml in (B)] from 30 min to 2 h. Results are shown as the ratio of acetylated histone to its respective total histone after normalizing immunoprecipitated chromatin in each sample to its respective total chromatin input. Values shown are the mean ± SEM from four independent experiments, each comparing one culture of Nfat5+/+ BMDM with Nfat5–/– BMDM from a littermate mouse. (C) Recruitment of p65 to the promoters of Nos2, Il6, and Tnf in Nfat5+/+ or Nfat5–/– BMDM pretreated (5 h) or not with the HDAC inhibitor TSA (500 nM) and then stimulated with 0.3 ng/ml LPS for 1 h. Values show the mean ± SEM from four to five independent experiments, each comparing one or two pairs of Nfat5+/+ and Nfat5–/– BMDM from littermate mice (*p < 0.05). (D) Abundance of histone H3, H3K4me3, H3K27me3, and H4K20me3 in the promoters of Nos2, Il6, and Tnf in wild-type BMDM in basal unstimulated conditions. Immunoprecipitated chromatin in each sample is shown relative to its respective total chromatin input. Values shown are the mean ± SEM from three independently performed experiments (*p < 0.05). (E) ChIP analysis of H3, H3K4me3, and H3K27me3 in the promoter and enhancer of Nos2 and the promoters of Il6 and Tnf in wild-type (Nfat5+/+) and NFAT5-deficient (Nfat5–/–) BMDM untreated (t = 0) or stimulated with LPS (0.3 ng/ml, 0.5–2 h). Immunoprecipitated chromatin in each sample was normalized to its respective total chromatin input. Values shown are the mean ± SEM from four independent experiments, each comparing one culture of Nfat5+/+ BMDM with Nfat5–/– BMDM from a littermate mouse (#p < 0.05 or §p < 0.05 between unstimulated and LPS-treated Nfat5+/+ (#) or Nfat5–/– (§) BMDM. *p < 0.05, **p < 0.01 between Nfat5+/+ and Nfat5–/– BMDM). (F) ChIP analysis of H4K20me3 in the promoters of Nos2, Il6, or Tnf in wild-type (Nfat5+/+) and NFAT5-deficient (Nfat5–/–) BMDM untreated (t = 0) or stimulated with LPS (0.3 ng/ml, 1–2 h). Immunoprecipitated chromatin in each sample is shown relative to its respective total chromatin input. Values shown are the mean ± SEM from three independent experiments, each comparing one culture of Nfat5+/+ BMDM with Nfat5–/– BMDM from a littermate mouse (#p < 0.05 between unstimulated and LPS-treated Nfat5+/+ BMDM). Statistical significance in (C)–(F) was assessed with a paired t test. The p values between 0.05 and 0.1 are indicated.