Fig. 5.

Normalization of Rasal1 promoter methylation upon de-methylating Hydralazine involves DNA hydroxylase Tet3. (A) As analyzed by qRT-PCR, Hydralazine treatment is associated with induction of DNA hydroxylase Tet3 in primary fibrotic fibroblast cultures (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) Panels display representative analysis of cells which had been transfected with scrambled siRNA (left) or with siRNAs targeting (from left to right) Tet1, -2, or -3. Whereas neither knockdown of Tet1 nor Tet2 affected the efficacy of Hydralazine to induce Rasal1 promoter de-methylation, Rasal1 hydroxymethylation, or Rasal1 carboxylation, depletion of Tet3 resulted in failure of Hydralazine to normalize Rasal1 methylation due to inefficacy of Rasal1 hydroxymethylation and Rasal1 carboxylation formation. (C) Fibrotic fibroblast cultures were subjected to vehicle buffer PBS, 5′-Azacytidine, or Hydralazine, Rasal1 mRNA expression levels were analyzed by qRT-PCR at indicated time points. Depletion of Tet1 and Tet2 did not affect the capacity of Hydralazine to restore Rasal1 mRNA expression. In contrast, Hydralazine no longer normalized decreased Rasal1 expression levels observed in fibrotic fibroblasts when Tet3 was depleted (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). (D) Consistent with failure of Hydralazine to normalize Rasal1 promoter methylation and mRNA expression after Tet3 depletion, Hydralazine no longer decreased the proliferative activity of fibrotic fibroblast cultures (experiments were replicated four times, data are presented as means ± s.e.m. *p < 0.05, n.s. no significance, values of p were calculated respective to two days vehicle-treated cultures).