Click it edu imaging kits

To test the cell proliferation, Click-iT EdU Imaging Kits (Beyotime, China) was used according to the manufacturer’s instructions, as previously studied [52 (link)]. Briefly, HeLa cells were planted at a density of 5 × 10⁴ cells in a 24-well plate 24 h before transfection. After transfection for 24 h, EdU was added to the medium according to the reagent instructions to make a final concentration of 10 μM. Then, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min. To detect EdU, the configured click reaction mixes were added to each well, incubated for 30 min at room temperature, and protected from light. Lastly, to detect the proportion of cell proliferation, nuclei were stained using Hoechst 33,342 and imaged with a Leica inverted fluorescence microscope (Leica, DMI3000B, Wetzlar, Germany).

DNA synthesis rate assessment was conducted utilizing Click-iT EDU Imaging Kits (Beyotime, Beijing, China), and experiments were finished with the method provided by the manufacturer. CCK-8 assay: 10 µl CCK-8 solution (Dojindo, Kumamoto, Japan) was added to 96-well plates at the time specified in the manufacturer’s instructions, which were incubated for 2 × 10³ cells, and then the absorbance was measured at 450 nm after continued incubation for 2 h at 37 °C. For the cloning experiments, crystal violet was utilized to stain the cell colonies, which were cultivated in 6-well plates for 14 days. All experiments were repeated three times.