Cell cycle assays by flow cytometry

For analysis of the cell cycle, SGC-7901 cells were harvested after 48 h transfection, prior to being washed with PBS and then fixed in ice-cold 70% ethanol at 4°C overnight. The next day, the cell were incubated with RNase A at 37°C for 30 min, and then stained with propidium iodide (PI; Sigma-Aldrich; Merck KGaA) at 4°C for 30 min in the dark, prior to the cells being analyzed by a flow cytometer using the CellQuest Pro software version 5.1 (BD Biosciences, Franklin Lakes, NJ, USA).