The cytotoxic effect of RNR inhibition via siRNA-mediated gene knockdown or 3-AP treatment is potentiated by VE-821. (A) AML-3 (red bars) and U937 cells (blue bars) were treated with RNR RRM1 siRNA (24 hours before dosing), 5 nM Gem, 15 µM HU, 50 nM Ara-C, or vehicle control in the presence of 1 µM VE-821 or DMSO control. Cell survival was determined after 48 hours, and the bar chart shows cell growth relative to vehicle controls. Numbers below bars represent the VE-821-induced potentiation factor (PF) and P values for each treatment determined by a 2-tailed Student t test. Western blotting for RRM1 was also performed 48 hours posttreatment (below the bar chart). (B) AML-3 (left) and U937 cells (right) with and without siRNA-mediated RRM1 knockdown were treated with 1 µM VE-821 (or DMSO control) for 48 hours, and western blotting was performed for RRM1 and pCHK1 (Ser345). (C) AML-2, AML-3, and U937 cells were treated with 5 µM 3-AP (or vehicle control) in the presence of 1 µM VE-821 (or DMSO control) for 2 hours, and western blotting was performed for pCHK1 (Ser345). (D) AML-2, AML-3, and U937 cells were treated with 3-AP alone (purple circles) or in combination with 1 µM VE-821 (red circles), and cell survival (relative to respective vehicle controls) was determined after 96 hours. PFs were calculated at the highest dose of 3-AP. For all western blots, GAPDH was used as a loading control, and for potentiation assays, data represent the mean ± SD of 3 independent experiments.