Colony formation assay

A total of 4000 cells were seeded on each well of a 6-well plate, and the cells were transfected with individual siRNA oligonucleotides by using Lipofectamine RNAiMAX (Invitrogen; Thermo Fisher Scientific Inc., Waltham, MA, USA) in the 6-well plate. After incubation at 37 °C for 3 days, the cultured medium was replaced with a new medium. The cells were allowed to incubate at 37 °C for 10 days. Cell culture plates containing colonies were fixed with 4% formaldehyde for 2 min and colonies were stained with crystal violet solution (containing 0.5% crystal violet, 5% formaldehyde, 50% ethanol, and 0.85% sodium chloride) for 2 h. Wells were rinsed with H₂O after air drying. The crystal violet staining of cells from each well was solubilized using 1 mL per well of 10% acetic acid, and the absorbance (optical density) of the solution was measured on a spectrophotometer at a wavelength of 595 nm.