Cell Counting Kit (CCK)-8 and Colony Formation Assays

The proliferation ability of the cells was evaluated by CCK-8 and colony formation assays. In the CCK-8 assay, 100 µl of suspension containing 1 × 10³ CRC cells was inoculated into each 96-well plate. Ten microliters of CCK-8 solution was added to each well, and the cells were incubated at 37°C for 4 h. The absorbance at 450 nm was measured at the indicated time points (24, 48, 72, and 96 h after transfection).

In the colony formation assay, 2 × 10³ CRC cells per well were seeded in a 6-well plate, cultured in 2 mL DMEM containing 10% FBS and 2 mL of RPMI 1640 medium containing 10% FBS, and incubated at 37°C and 5% CO₂ for 24 h. The medium was changed every 2 days, and the culture was terminated on the 12^th day. The cells were immobilized with 4% paraformaldehyde for 30 min, purified twice with phosphate-buffered saline (PBS), and stained with 2.5% crystal violet. Subsequently, live colonies were counted.