Clone formation assay

Cells were seeded at 500 cells/well in six-well plates and pipetted several times, so that most cells were in single-cell forms. After 3 days of incubation, the cells were treated with DMSO (vehicle control) or GSK650394 at various concentrations (2.5–10 μM), and the covering medium was changed every 2 or 3 days during culture. After 10–14 days, when the cells grew to visible colonies, the medium was removed, and the cells were washed with PBS twice. After being fixed with 4% paraformaldehyde for 15 min and washed with PBS, the cells were stained with 0.1% crystal violet for 10 min. Finally, the excess dye was washed away with PBS, and then, cells were visualised using an ordinary optical microscope (with × 4 objective). The plates were prepared in triplicate, and 10 fields for each well were chosen randomly. Then, the number of colonies in each field was counted, only colonies containing more than approximately 50 cells were counted, and the size of the colonies was measured.