Hoechest staining solution

After CC cells were seeded in 96-well plates at a density of 2 × 10³ cells/well and cultured for 24 h, the BrdU kit (BD Pharmingen, San Diego, CA, USA) was added into each well (final concentration: 10 µM), and the culture was continued for 8 h. Subsequently, the medium was removed, and cells were fixed with 4% paraformaldehyde for 30 min and then incubated with anti-BrdU antibody (Beyotime, Shanghai, China) for 1 h at room temperature. After the cells were rinsed with phosphate buffer saline (PBS) for 3 times, Hoechest staining solution (Beyotime, Shanghai, China) was used for marking cell nuclei. Three fields were randomly selected under a fluorescence microscope. Cell proliferation rate = BrdU positive cells / Hoechst positive cells × 100%.

The transfected GC cells in each group were transferred onto coverslips (Beyotime, Shanghai, China) and then cultured for 12 h. Subsequently, the cells were incubated BrdU solution (Beyotime, Shanghai, China) for 6 h. The GC cells, after the medium was discarded, were fixed with 4% paraformaldehyde for 30 min, and incubated with anti-BrdU antibody (Beyotime, Shanghai, China) at ambient temperature for 1 h, and then rinsed with phosphate buffer saline (PBS). Subsequently, the nuclei were stained employing Hoechest staining solution (Beyotime, Shanghai, China). After the cells were washed by PBS again, the numbers of BrdU and Hoechst positive cells were counted.