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. Author manuscript; available in PMC: 2015 Feb 23.
Published in final edited form as: EBioMedicine. 2015 Jan;2(1):19–36. doi: 10.1016/j.ebiom.2014.11.005

Fig. 4.

Fig. 4

Hydralazine-induced de-methylation of Rasal1 promoter is mediated through active mechanisms in renal fibroblasts and not exclusively dependent on Dnmt1 inhibition. (A) Analyzed by qRT-PCR, mRNA expression levels of DNA methyltransferases Dnmt1 and Dnmt3a were normalized after treatment with 5′-Azacytidine and Hydralazine (experiments were done in triplicate, data are presented as means ± s.e.m. *p < 0.05, n.s. no significance, values of p were calculated respective to vehicle-treated fibrotic cultures). (B) The top pictures show virtual gel images of Rasal1 PCR products of immunoprecipitated DNA, the bottom pictures show PCR products of input DNA as controls for equal loading in MeDIP, hMeDIP, and CaDIP. Panels display representative analysis of cells which had been transfected with scrambled siRNA (left) or with siRNAs targeting (from left to right) Dnmt1, -3a, or 3b. Knockdown of Dnmts did not mediate rapid Rasal1 de-methylation as observed upon treatment with Hydralazine suggesting that Rasal1 de-methylation upon Hydralazine exposure involves mechanisms of active DNA de-methylation. (C) Fibrotic fibroblast cultures were subjected to vehicle buffer, 5′-Azacytidine or Hydralazine, Rasal1 mRNA expression levels were analyzed by qRT-PCR at indicated time points. Decreased Rasal1 mRNA expression levels observed in fibrotic fibroblast cultures were restored after treatment with de-methylating 5′-Azacytidine or Hydralazine. Knockdown of Dnmts did not affect Rasal1 mRNA expression levels (experiments were done in triplicate, data are presented as means ± s.e.m. *p < 0.05, values of p were calculated respective to two days vehicle-treated fibrotic fibroblast cultures). (D) Depletion of Dnmt1, -3a, or -3b did not affect proliferative activity of fibrotic fibroblasts, 5′-Azacytidine and Hydralazine were still capable to normalize intrinsic proliferative activity (experiments were replicated four times, data are presented as means ± s.e.m. *p < 0.05, values of p were calculated respective to two days vehicle-treated cultures).