Fig. 6.

Normalization of Rasal1 methylation by Hydralazine involves DNA glycosylase Tdg. (A) As analyzed by qRT-PCR, Hydralazine administration is associated with induction of DNA glycosylases Tdg and Smug 1 (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) Cells had been transfected with scrambled siRNA (left) or with siRNAs targeting Tdg (middle) or Smug1 (right). Knockdown of Tdg resulted in impaired Rasal1 promoter de-methylation upon Hydralazine treatment whereas depletion of Smug1 did not affect normalization of Rasal1 promoter methylation. (C) Whereas knockdown of Smug1 did not affect normalization of Rasal1 mRNA expression upon Hydralazine treatment, Hydralazine failed to restore Rasal1 expression levels when Tdg 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 insufficient normalization of Rasal1 promoter methylation and mRNA expression after Tdg 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).