Cell proliferation assay

For the MTT assay, the cell suspension was seeded in each well of 96-well plates. After 12 h, attached cells were treated with various doses of 5-FU or/and IL-1RA. The cells were incubated in 4.5% CO₂ at 37 °C for 1, 2, 3, 4, and 5 d, 10 µL of MTT solution (5 mg/mL) was added to crystallize the cells for 4 h, and 150 µL of DMSO was added for 10 min to oscillate the cells. The absorbance value was measured at 490 nm. In the colony formation assay, DMEM-diluted cell suspension was inoculated in a 6-well culture dish containing 10 mL of 37 °C incubation medium at a density of 300 cells per well. After 12 h, attached cells were treated with various doses of 5-FU or/and IL-1RA. The cells were incubated in a cell incubator for 2 wk, and then, the colonies were immobilized with formalin (Sigma-Aldrich) within 30 min and stained with crystal violet (Sigma-Aldrich) within 1 h.