5-azaC down-regulates RRM2 expression in vitro. (A) The RRM2 protein level was reduced by 5-azaC in a dose- and time-dependent manner in MV4-11 and K562 cells. Densitometry was performed to quantify each lane and the ratio of RRM2 over the loading control GAPDH is presented under each blot. (B) Representative densitometry plots. (C) 5-azaC down-regulates RRM2 mRNA in a dose-dependent manner. Cells were treated with serial concentrations of 5-azaC for 24 hours and collected for analysis. (D) 5-azaC down-regulates RRM2 mRNA in a time-dependent manner. MV4-11 cells were treated with 5μM 5-azaC for the indicated times and the RRM2 mRNA level was quantified. The RRM2 mRNA levels were normalized by an internal control, abl. Data are presented as the percentage of the untreated controls. *P < .05 and **P < .01 compared with the controls. (E) Decitabine (DAC) has minimal effects on RRM2 protein expressions. (F-G) Effects of 2 RRM2 inhibitors, Triapine (F) and GTI-2040 (G), on the RRM2 protein level. (H) Comparative plot of RRM2 protein levels in MV4-11 cells after 5-azaC, Triapine, or GTI-2040 treatment.