Cell cycle analysis

To investigate the mechanisms underlying the effects of USP39 on cell growth, cell cycle distribution was examined by flow cytometry and propidium iodide (PI) staining. SW1116 (7×10⁴ cells/well) and HCT116 (2×10⁵ cells/well) cells were harvested at 7 days and 5 days after infection, respectively. Cells were fixed in 70% ice-cold ethanol for 4 h and washed twice with cold PBS. The fixed cells were stained with PI and detected by a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) according to the manufacturer's protocol. The results were analyzed using FlowJo software version 7.6.2 (FlowJo LLC, Ashland, OR, USA).