B16/F10 cells were seeded in 10 cm dishes at a density of 1 × 106 cells and cultured for 12-16 hrs in an incubator. After cell fulling to 60%-70% of dishes, the indicating drugs were given to cells for 6, 12, 24 and 48 hrs. After treatment, the cells were harvested and stained with PI (40 μg/ml) in PBS containing 100 μg/ml RNase overnight at 4°C. Cell cycle distribution was evaluated in independent tests for three times. The detailed analyzed method we utilized in this experiment was described as the following. We selected a fixed area of sub-G1, G0/G1, S, and G2/M phase in each group. All cells were divided into two groups, and one was the living cell population to establish the purpose of observing drug-induced changes of the cell population in cell cycle distribution. The calculated formula of cell cycle distribution was the indicated cell population (G0/G1, S, or G2/M phase) / total alive cells × 100%. Another was a dead cell population to evaluate drug-induced cell death. The area of sub-G1 phase was set below 160 of FL2-A and the calculated formula of sub-G1 phase was sub-G1 cell population / total cell population containing alive and dead cells × 100%. Cell cycle distribution was determined using a FACSCalibur (Franklin Lakes, NJ, USA) and analyzed using FlowJo 7.6.1 (Ashland, Oregon, USA).

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