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. 2018 Nov 2;3(21):e95625. doi: 10.1172/jci.insight.95625

Figure 4. Effect of dexamethasone (Dex) on Agtr1a mRNA and DNA methylation of the Agtr1a promoter in the immortal mouse fetal PVN neuronal cell line, N39.

Figure 4

(A) Real-time PCR of Dnmt1, Dnmt3a, Agtr1a, and Agtr1b mRNA following treatment for 6 days with vehicle (n = 6) or Dex (n = 6). (B) Quantitation of DNA methylation (by MeDIP) at the Agtr1a gene +15/+251 from the transcription start site in vehicle-treated or Dex-treated cells (each n = 8). (C) ChIP assays of DNMT1 (left) and DNMT3a (right) binding to the site +15/+251 relative to the TSS in vehicle-treated or Dex-treated cells (n = 9 and 10 in DNMT1, and n = 7 and 10 in DNMT3a, respectively). (D) siRNA against DNMT3a (n = 10) significantly decreased DNA methylation of Agtr1a relative to scrambled siRNA (n = 11). (E) Effect of Dex treatment on Agtr1a mRNA expression in cells transfected with siDnmt3a (control and Dex, n = 12 and 11, respectively) or scrambled RNA (control and Dex, n = 10 and 8, respectively). (A–C) Filled circles, vehicle-treated cells; open circles, Dex-treated cells. (D–E) Filled circle, cells treated with scrambled RNA; open circles, cells treated with siRNA against Dnmt3a. Data represent means ± SEM. *P < 0.05 (A–D) t test; (E) Kruskal-Wallis test with post hoc Dunn’s multiple comparison.