The EdU Proliferation Assay

It was performed according to the manufacturer's instructions (RiboBio, Guangzhou, China). After transfection for 24 h, Caki-1 and SN12-PM6 cells were cultured in DMEM containing EdU at 37°C for 6 h. Then, cells were fixed with 4% formaldehyde for 20 min, followed by treatment with glycine for 5 min. We used treatment with 0.5% Triton X-100 at 28°C for 10 min to permeabilize the cell membranes. After washing twice, each well was treated with 200 μL of 1x Apollo reaction cocktail for 20 min. Subsequently, nuclear DNA was stained with Hoechst and imaged using fluorescence-based microscopy (Motic, Hongkong, China).