Cell cycle evaluation by flow cytometry

Transfected cells were collected and fixed in 4 ml of ice-cold 70% ethanol. After cell fixation at 4°C overnight in ethanol, the cells were centrifuged at 1,800×g for 5 min, then washed twice with ice-cold PBS, resuspended in 1 ml of propidium iodide (PI) staining solution (Beyotime, Shanghai, China) in the dark at 37°C for 10 min. A FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) was used to analyze the distribution of cells in different phases of the cell cycle, including G0–G1, S, and G2-M phases. The experiments were performed in triplicate.