Cells were synchronized by serum starvation for 18 hours and then released for 10 hours. Cells (1 × 105 cells per sample) were fixed in cold 70% ethanol at 4°C overnight and permeabilized with 0.1% Triton X-100. To analyze DNA content, samples were treated with propidium iodide (0.05 mg/ml) (P4170; Sigma-Aldrich) and RNase A (ribonuclease A) solution (0.1 mg/ml) (R4642; Sigma-Aldrich) at room temperature for 30 min and were analyzed immediately by flow cytometry using a BD FACSCalibur flow cytometer. Data were gated by using Kaluza analysis software.

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