Excess dNTP reverses synergy between MI-63 and dFdC. (A) Granta-519 cells were incubated with vehicle, 5μM MI-63, 10nM dFdC (GEM), or both agents simultaneously for 24 hours, either without or with exogenous dNTPs at 50μM. Cell death was then determined by flow cytometry using annexin-V and TO-PRO-3 staining relative to the vehicle control. Each panel provides representative data from 1 of 3 independent experiments. An unpaired t test was performed comparing cells to which dNTPs had been added to those exposed to drug alone; *P < .05. (B) Cellular lysates were probed for PARP, HDM-2, p53, RRM2, RRM2B, and p21, as well as β-actin as a loading control. (C) REC-1 cells were infected with Lentiviral particles carrying a scrambled sequence shRNA or an shRNA targeting RRM2, and stable cell lines were generated by drug selection. Cellular lysates were then probed for their content of RRM2, RRM2B, and β-actin as a loading control. (D) REC-1 shRNA cells were incubated with vehicle, 5μM MI-63, 10nM dFdC, or both agents simultaneously for 24 hours, and the proportion of cells undergoing apoptosis was determined by flow cytometry using annexin-V and TO-PRO-3. Statistically significant differences are defined as *P < .05.