Cell Viability and Proliferation Assay

Cell viability was evaluated using CCK8 (Beyotime, Jiangsu, China). Cells (2 × 10³) were seeded in 96-well plates by different treatment, and cultured for different time points. CCK8 solution (10 μl) was added to each well, and the plates were incubated at 37°C for 1 hour. Absorbance was measured at 450 nm on a microplate reader (Bioteck). DMEM containing 10% CCK8 was used as a control. Proliferating cell count was measured using the Cell-Light EdU DNA Cell Proliferation Kit (RIBOBio Co, Guangzhou, China). The cells were seeded in 96-well culture plates and exposed to media with or without plumbagin. 2000 cells/well were treated with 50 μmol/L of EdU for 2 h at 37 °C. After being fixed with 4% paraformaldehyde for 15 min, the cells were baptized with 0.5% Triton X-100 for 30 minutes and rinsed with PBS three times. Thereafter, the cells were exposed to 100 μL/well of 1×Apollo® reaction cocktail for 30 minutes and incubated with 5 μg/mL of Hoechst 33342 to stain the cell nuclei for 30 minutes. Images were captured using a fluorescent microscope (Olympus, Tokyo, Japan). Experiments were repeated at least three times.