Clonogenic survival assay

Cell proliferation ability was measured using a colony formation assay. Approximately 500 cells were seeded in each well on a 12-well plate as a single cell suspension. After 24 h, different concentrations of arenobufagin (0, 5, 10 and 50 nM) were added and the cells continued to be maintained at 37°C with a humidified atmosphere of 5% CO₂ for 15–20 days. Visible colonies were then stained with crystal violet and manually counted.