Colony Formation Assay for A549 and H1299 Cells

The colony formation assay was performed as previously described (Yu et al., 2020 (link)). Briefly, A549 and H1299 cells were seeded in 6-well plates at a density of 500 cells/well and allowed to adhere for approximately 12 h. The cells were then treated with DMEM containing 25 μg/mL EF40 for 6 h, followed by culturing in normal medium for 10 days. Cells treated with normal medium were used as the controls. After treatment, cells were fixed with a methanol/acetone (v/v, 1:1) mixture for 10 min and stained overnight with 0.5% crystal violet solution (Wako). The number of colonies was counted, and the mean number of colonies was calculated from three independent experiments.