Cells were seeded at the same density in 6-well plate and infected with lenti-viral shRNA, and medium was replaced 24 h later. Cells were then cultured in a humidified, 5% CO2 atmosphere incubator at 37 °C for 2-3 weeks until colonies developed. The cells were fixed in fixative solution (acetic acid: methanol =1:3) for 15 min and stained with 0.1% crystal violet for 15 min. For quantification, the crystal violet dye was released into 10% acetic acid and data was recorded at wavelength 590 nm.