Cell cycle analysis.

The cell cycle distribution was analyzed using flow cytometry. Before analysis, the cells were serum deprived for 24 h to synchronize the cell cycle or were synchronized by use of a double thymidine block, and then the cells were incubated with α-MEM containing 10% FBS for 24 h after serum deprivation. The cells were trypsinized, rinsed with PBS, and fixed with 70% ice-cold ethanol at 4°C or −20°C overnight. Before analysis, the cells were washed with PBS and resuspended in 0.05 mg/ml propidium iodide (PI; Sigma, St. Louis, MO) solution containing RNase A (0.02 mg/ml). The cells were incubated for 30 min in the dark and analyzed by flow cytometry (BD Biosciences). The results for at least 1×10⁴ cells were recorded for each sample, and the data were analyzed using Cell Quest software (BD Biosciences).