5-ethynyl-2′-deoxyuridine (EdU) labeling assay

Well-growing passage 3 cells were collected, and the DNA replication ability of cells was detected using a cell-light EdU labeling kit (Cat. No. C10310-1, RiboBio Co., Guangzhou, Guangdong China) as per the manufacturer’s instructions. In brief, cells were seeded onto 24-well plates at 2×10⁵ cells per well and incubated in 10% FBS-supplemented medium for 24 h. Next, the cells were treated with diluted EdU labeling reagent (1: 1000, RiboBio) for 6 h, and then the cells were fixed in 4% paraformaldehyde for 30 min and immersed in 2 μmg/mL glycine solution for 5 min. After that, the cells were incubated with 0.5% TritonX-100 (#A110694, Sangon) in phosphate-buffered saline (PBS) at room temperature for 20 min. The labeled cells were observed under a fluorescence microscope (Eclipse TS100, Nikon). Nuclei stained by 4′, 6-diamidino-2-phenylindole (DAPI) were observed by ultraviolet photoluminescence (wavelength=425 nm) while the EdU-labeled cells were observed using green photoluminescence (wavelength=590 nm), and the ratio of EdU-positive cells to total cells was evaluated by Image J software.