Colony formation assay

Cells in the logarithmic growth phase were plated at 2,000 cells/well in a 6-well plate. Before seeding, cells in the HIFU and HIFU+bufalin groups were treated with HIFU. After 12 hours, bufalin was added into the medium at a final concentration of 20 nM in the bufalin and HIFU+bufalin groups. Fresh medium was replaced every 2 days. After 2 weeks, the cells were fixed and stained with 0.5% crystal violet for 20 minutes at room temperature. Excess crystal violet was removed by three washes with water, the plates were dried, and images were captured by using a microscope. The number of cell colonies was calculated by using ImageJ software. Each experiment was performed in triplicate.