2.6. Cell Cycle Kit Assay

When cells grew to 80% confluence, they were re-seeded onto 6-well plates (1 mL/well). Transfection was performed under 60% cell confluence, followed by 24h incubation. Cells were digested with 0.25% trypsin and centrifuged at 1000× g for 5 min with 1 mL PBS. Each well was added 1 mL pre-cooled 70% ethanol, mixed by pipetting, and was fixed at 4 °C for 24 h. Then, cells were rinsed by pre-cooled PBS and centrifuged. Next, a solution of propidium iodide was prepared according to the Beyotime Cell Cycle Kit (Beyotime, Shanghai, China). To each tube of cells was added 0.5 mL staining solution, incubated at 37 °C for 30 min in the dark, and detected by flow cytometric.