BrdU incorporation assay

A BrdU cell proliferation ELISA kit (Abcam, Cambridge, MA, USA) was used to quantify cells in DNA synthesis. Briefly, cells were incubated with fresh medium containing BrdU solution for 12 h at 37°C. After removing the medium and being washed in PBS for 5 min at room temperature (RT), cells were fixed in 4% paraformaldehyde solution for 10 min at RT and incubated with primary BrdU antibody for 1 h at RT. Cells were subsequently incubated with the secondary antibody for 30 min at RT. Following incubation of cells with TMD and stop solution, the absorbance was determined at 450 nm using a microplate reader (Thermo Fisher Scientific, Inc., USA).