Cell cycle analysis

The NCOA5-silenced SW620 and NCOA5-overexpressed SW480 CRC cells and the corresponding control cells were cultured and collected. Then the cells were washed with phosphate-buffered saline (PBS) and fixed in ice-cold 70% ethanol at 4 °C overnight. They were subsequently washed with PBS, treated with 500 U/ml RNase A (Sigma, St Louis, MO, USA) at 37 °C for 30 min and stained with 50 μg/ml propidium iodide (PI) (Sigma, St Louis, MO, USA) in the dark for 30 min. Finally, the cells were washed and analyzed for cell cycle by FACSCalibur flow cytometry (BD, Franklin Lakes, NJ, USA).