RRM2 is involved in the conversion of 5-azaC to DAC in MV4-11 cells. (A-C) 5-azaC rapidly converts to DAC and DAC-TP, which diminishes with time. Knock-down of RRM2 expression prevents 5-azaC conversion to DAC (A) and DAC-TP (B) and associated events (C). MV4-11 cells were transfected with siRRM2 or mock-transfected for 24 hours, followed by treatment with 10μM 5-azaC for 2-28 hours. The arrows indicate sequences of fresh addition of 5-azaC. The intracellular DAC and DAC-TP were extracted and quantified by LC-MS/MS. Cells treated with 10μM DAC for 2 hours serve as a positive control. Data are presented as a percentage of the positive controls. Knock-down of RRM2 expression inhibits 5-azaC–induced down-regulation of DNMT1 and DNMT3a (C). Western blot analysis shows that 5-azaC alone time-dependently decreases the protein levels of DNMT1 and DNMT3a, whereas RRM2 depletion by siRRM2 abolishes its effects. Experiments were repeated and the representative results are presented. (D-E) Conversion of 5-azaC to DAC is reduced by UCK inhibitors. MV4-11 cells were treated with 10μM 5-azaC for 2 or 4 hours. UCK was inhibited competitively by its natural substrates, cytidine or uridine. Intracellular DAC (D) and DAC-TP (E) levels were measured. Cells treated with 10μM DAC for 2 or 4 hours serve as positive controls. (F) Stability of DAC. MV4-11 cells in PBS were heat-inactivated at 100°C for 6 minutes and then exposed to 10μM 5-azaC or DAC for 2 or 4 hours at 37°C.