The cell cycle was examined by flow cytometric analysis with propidium iodide (PI)/RNase staining kit (Beyotime, China). Cell suspensions at a density of 3 × 105 cells/well were seeded in 6-well plates and incubated overnight. After cycle synchronization by serum starvation for 24 h, cells were cultured in regular DMEM for 48 h. Then, cells were washed in cold PBS and centrifuged at 1000×g for 5 min, and resuspended in ice-cold 70% ethanol for at least 2 h on ice. Centrifuged, decant and washed again in cold PBS. About 1 × 106 cells were resuspended in 0.5 ml working solution per sample (500 µl of assay buffer mixed 25 µl of PI solution and 2.5 µl RNase solution), and kept for 30 min at 37 °C and 4 °C in the dark, respectively.
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