Detection of cell proliferation, cell cycle, and cell apoptosis

MTT assay, plate clone formation assay, Edu assay, flow cytometry for cell cycle, and cell apoptosis were conducted according to the protocols described in our previous studies^25,26. Briefly, with respect to MTT assay, proliferation of cells was measured by MTT solution (5 mg/ml; Beyotime Institute of Biotechnology, Shanghai, China) at 24, 48, and 72 h, respectively. With respect to plate clone formation assay, 1 × 10³ cells were seeded in 6-well plates. The cells were mixed and then cultured for 2 weeks in culture medium with 10% FBS. Clusters containing ≥30 cells were counted as a single colony. With respect to Edu assay, Cell-Light™ EdU Apollo®488 In Vitro Imaging Kit (#C10310-3, RiboBio Co., LTD, Guangzhou, China) was used. With respect to flow cytometry for cell cycle and cell apoptosis, PE Annexin V Apoptosis Detection Kit I (#559763, Becton Dickinson bioscience, San Jose, CA, USA) and PI/RNase Staining Buffer (#550825, Becton Dickinson bioscience, San Jose, CA, USA) were used. All of the above experiments were performed according to the manufacturer’s protocols.