Colony formation and soft agar assays

For colony formation assays, HCT116 and SW480 cells were transfected with siRNAs of CDC27 or negative control siRNA, and DLD1 cells were transfected with CDC27-carring plasmids or empty vector. Cells were plated into six-well plates at 48 h after transfection. After incubating at 37 °C for 14 days, visible colonies were fixed and stained with 0.5% crystal violet in methanol. For the soft agar assay, the cells in single-cell suspension were plated in 0.3% agarose over a 0.6% agarose bottom layer in six-well plates and incubated for 14 days. The number of colonies containing more than 50 cells was counted.