Hoechst33342 solution

EdU (KeyGEN BioTECH, China) was added to the cell culture medium to a final concentration of 20 μM before incubation at 37 °C for 4 h. The cells were then fixed with 4% paraformaldehyde (Biosharp, USA) for 20 min at room temperature, washed with PBS, and treated with 0.5% Triton X-100 (Sigma, Germany) for 20 min at room temperature. Next, the reaction buffer and KFlour488-azide (KeyGEN BioTECH, China) were mixed and incubated with the cells at room temperature protected from light for 30 min. Lastly, the cells were washed with PBS twice and incubated with Hoechst33342 solution (KeyGEN BioTECH, China) at room temperature in the dark for 30 min.

EdU staining was performed using kFluor647-EdU cell proliferation assay kit (Keygen Biotech, Nanjing, China). Forty-eight h after transfection, cells were maintained in 10 μM EdU (Keygen Biotech) for 12 h after PBS washing. Then, fixation and penetration of cells were performed, followed by cell incubation in Click-iT reaction mixture (Keygen Biotech). Hoechst 33,342 solution (Keygen Biotech) was used for DNA counter-staining. After mounting, the slices were observed and pictured under a microscope (Olympus, Tokyo, Japan) at 400x magnification.

After treatment, cells in six-well plates were incubated with Hoechst 33342 solution (KGA212; KeyGEN BioTECH) at 37°C for 10 min. Next, they were washed with staining buffer followed by incubation with PI solution at room temperature for 10 min. After PI incubation, the stained cells were observed and photographed using a fluorescence microscope (Leica).