Fig. 7.

Aldo-induced reprogramming of methylation of the R3 (−57/+438) subregion of the αENaC promoter. The time course of Aldo-induced changes in methylation marks and modifiers is shown. mIMCD3 cells were cultured in DMEM-F-12 plus 10% charcoal-stripped serum for 20 h and then treated with vehicle or 1 μM Aldo for the indicated time periods. Cells were then harvested and subject to DNA immunoprecipitation/qPCR assays with antibodies specific for 5mC or 5-hydroxymethylcytosine (5hmC; A) and ChIP/qPCR for DNMT3b (B) and ten-eleven translocation (Tet)1, 2, and 3 (C). Specific primers were used to amplify the R3 subregion of αENaC. Data from Aldo-treated samples were normalized to the vehicle time-control values, which did not significantly vary over the time period. Data in A–C are presented as fold changes of the indicated time relative to their value at time 0. Error bars indicate ±SEs; n = 3. *P < 0.05 vs. the corresponding value at time 0. D: mIMCD3 cells were transiently transfected with control siRNA or with Tet2-specific siRNA. Cells were then treated with vehicle or Aldo for 24 h, and αENaC mRNA, which was normalized to β-actin mRNA, was measured. The value of Aldo-treated control siRNA-transfected cells was set at 1, and the other data were normalized to it. n = 6. *P < 0.05 vs. control siRNA.