Cell-Cycle Analysis

To understand the antiproliferative efficacy of the combination treatments, cells were analyzed for cell-cycle analysis at 1, 3, 5, or 8 days post treatment, as described previously [10]. Briefly, 1 × 10⁶ cells were seeded in a 100-mm-diameter cell-culture dish (Becton Dickinson, San Jose, CA). For 8-day treatment, the initial cell seeding was 0.5 × 10⁶ cells. Cells were allowed to attach for 24 h prior to treatment. At different time points post treatment, cells were harvested by trypsinization and centrifuged at 1300 rpm for 3 min at 4 °C (Sorvall Legend RT centrifuge, Thermo Electron Corp., Waltham, MA). The supernatant was decanted, the cell pellet was washed twice with ice-cold 1×DPBS (pH 7.4), and then the pellet was resuspended in a solution (12.5 mg propidium iodide [Promega, Madison, WI], 250 mg sodium-citrate, and 250 μL Triton™ X-100 in 250 mL of water). The cells in the propidium iodide solution were then incubated for 2 h in the dark in a cold room and then analyzed by flow cytometry (FACScan flow cytometer, BD Biosciences, San Jose, CA). ModFit LT software (Verity Software House, Inc., Topsham, ME) was used for data analysis.