dFdC and MI-63 enhance cell death. (A) REC-1, Granta-519, and JVM-2, and the mutp53 MCL cell line JeKo-1, were treated for 24 hours with vehicle, 5μM MI-63, 10nM dFdC (GEM), or both. Flow cytometric analysis was then performed after staining with annexin-V and TO-PRO-3, from which the proportion of cells undergoing apoptosis was calculated and normalized to the vehicle control group. Values represent the mean ± SE from 3 independent experiments. An unpaired t test was performed to evaluate for significance; *P < .01 relative to MI-63 alone; #P < .01 relative to dFdC alone. (B) Granta-519 cells were incubated simultaneously with single agent MI-63 or dFdC for 72 hours. In parallel, cells were exposed either first to MI-63 for 24 hours followed by dFdC and MI-63 for 48 hours or to dFdC first for 24 hours followed later by MI-63 and dFdC for 48 hours. Cell viability was determined using the WST-1 reagent, and results are expressed as the percentage viability relative to the vehicle control, which was arbitrarily set at 100%. The presence of synergistic interactions was determined by calculation of the CI from the cell viabilities calculated across a serial dilution range of MI-63 or dFdC (Table 2). Each panel provides representative data from 1 of 3 independent experiments. (C) Protein levels of HDM-2, p53, RRM1, RRM2, RRM2B, and p21, and β-actin as a loading control, were determined by Western blotting of cellular lysates.