Synergistic activity of MI-63 and dFdC in MCL patient samples. (A) MCL cells were purified from the peripheral blood of patients with circulating neoplastic cells using magnetic-activated cell sorting and CD19 microbeads. These cells were then either exposed to 5μM MI-63, 10nM dFdC, or both agents simultaneously for 24 hours. Cell death was determined by flow cytometry using annexin-V and TO-PRO-3 staining relative to the vehicle-treated control, and REC-1 cells were included as an additional control. Each panel provides representative data from 1 of 3 independent experiments, and *P < .05 denote significance relative to MI-63 alone, and #P < .05 denote significance relative to dFdC alone. (B) Aliquots of each of the primary samples analyzed in panel A also were subjected to RNA extraction, cDNA was synthesized, and the levels of RRM1, dCk, hENT-1, and RRM2 were measured by quantitative real-time PCR using the ΔΔCT method with the JVM-2 cell line used as a relative calibrator. The transcript level in REC-1 cells also was measured as a control and is plotted on a separate scale because of their high expression of RRM1, dCK, and hENT-1. (C) Real-time PCR analysis of RRM2 transcript levels in MCL patient samples is shown, along with REC-1 as a cell line control.