Cell viability assay

Cells were seeded in a 96-well plate at a density of 5,000 cells/well for MTT assays. Treatments with different concentrations of repertaxin (dissolved in 0.9% saline) alone (0.025, 0.25, 2.5, 25 and 50 μg/ml) (Sigma-Aldrich), 5-FU alone (0.01, 0.1, 1, 10 and 20 μg/ml) (Shanghai Xudong Haipu Pharmaceutical, Co., Ltd., Shanghai, China), and repertaxin (0.025, 0.25, 2.5, 25 and 50 μg/ml) in combination with 5-FU (0.01, 0.1, 1, 10 and 20 μg/ml) were administered 12 h after seeding. Cell viability was determined on days 1, 2, 3, 4 and 5 after treatment by addition of 20 μl MTT solution (5 mg/ml in PBS) (Sigma-Aldrich). Cells were incubated in MTT solution for 4 h at 37°C. Next, the culture medium was removed and replaced with 150 μl DMSO to dissolve the crystals. Samples were incubated at room temperature for 10 min, and then absorbance was measured at 568 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).