Edu reagent

Cells seeded in 96-well plates were incubated with 100 μL EdU reagent (Solarbio) for 2 h. The cells were washed with phosphate-buffered saline (PBS) and stained with Apollo reagent. Next, the cells were stained with methanol, fixed, permeated, and then cultured with 100 μL 4′,6-diamidino-2-phenylindole (DAPI) reaction solution (Sigma–Aldrich, Merck KGaA, Darmstadt, Germany). Positive EdU labeling, namely, the viability of cells, was determined under a fluorescence microscope (Thermo Fisher Scientific).

The transfected cells were cultured in 96-well plates in triplicate (4 × 10⁴ cells per well). After 48 h, each well was loaded with 100 μL EdU reagent (Solarbio Science & Technology, Beijing, China) for 2 h. Thereafter, cells were further incubated with cell fixing reagent (100 μL per well), 2 mg/mL glycine, and penetrant (100 μL per well, 0.5% Triton X-100-supplemented phosphate-buffered saline at 22–25°C for 30 min. After that, cells were stained with 1× Apollo and 1 × Hoechst 33,342 solutions (100 μL per well), and then incubated with the anti-fluorescence quenching solution (100 μL per well). The labeling was observed under a microscope with three random fields included. The EdU-positive cells were stained in red while total cells were in blue. The EdU-positive rate (%) was calculated as follow: rate = number of EdU-positive cells/number of total cells × 100% [15 (link)].