Cck 8

Cell counting kit-8 (CCK-8) (E-CK-A361; Elabscience, USA) was used to conduct cell viability assays. HCT116 and HT-29 cells were seeded in 6-well plates at 2 × 10⁵ cells/well and incubated for 24 h. After siRNA transfection for 48 h, CCK8 solution and RPMI-1640 medium with 10% FBS were added to each well and incubated with 5% CO₂ at 37°C for 2 min or 5 min. The absorbance was assessed using a microplate reader at 450 nm. For crystal violet staining, the cells were fixed with methanol for 5 min and stained with 0.1% crystal violet after siRNA transfection for 48 h.

To determine cell viability, cell counting kit‐8 (CCK‐8, Elabscience) was used. A total of 3 × 10³ cells were seeded and cultured in 96‐well microplates using 100 μL of medium per well. After culturing for 24 h, 10 L of CCK‐8 reagent was added to each well, and the cells were cultured for 90 min. In all experiments, three replicates were performed. Wells without cells were used as blanks, and cell proliferation was measured by absorbance. Absorbance analysis was performed at 450 nm using a SpectraMax Absorbance Reader (Molecular Devices). In the colony‐forming assay, cells were seeded into six‐well plates at a density of 1 × 10³ cells per well in DMEM with 10% FBS and cultured for 14 days to allow colony formation. Then, 4% polyformaldehyde was applied at room temperature to the plate after it was washed in cold PBS. In the next step, they were dyed at room temperature for 30 min with 1% crystal violet. Each experiment was performed three times, counting colonies over 100 cells by using ImageJ.