The effect of adenovirus-transduced DNMT genes on DNA synthesis and cyclin A mRNA levels in Hcy-treated HUVECs. HUVECs were infected with control adenovirus (Ad-CT) (A), adenovirus expressing DNMT1 (Adv-DNMT1) (B,D), and adenovirus expressing DNMT3 (Adv-DNMT3) (C) at the indicated MOI for 24 hours, and then treated with 50 μM DL-Hcy in control medium for another 24 hours. (A-C) Thymidine uptake. Cells were metabolically labeled with 1 μCi (0.037 MBq)/mL [³H]-thymidine during the last 3 hours. [³H]-thymidine incorporation was measured in a liquid scintillation counter. Values represent mean (± SD) from 3 separate experiments with triplicates (n = 9). *P < .01 versus non-Hcy and nonviral control. #P < .01 versus Hcy-treated and non–viral-transduced group. Protein levels of DNMT1 and DNMT3 were examined by Western blotting with antibodies against DNMT1 or DNMT3, and reblotted with β-actin antibody. (D) mRNA levels: HUVECs were infected with Adv-DNMT1 for 24 hours and then treated with DL-Hcy in control medium for another 48 hours. Total cellular RNA (10 μg) was extracted and used for Northern analysis. The blot was hybridized with the probes of human cyclin A and D1, and subsequently with 18S RNA.