Colony formation assay

HCC cells (5×10² cells per well) were seeded into a 6-well plate and incubated under normal culture conditions for 24 h. The cells were treated with a series of cisplatin concentrations for 2 weeks. Cells were fixed with ice-cold methanol at -20°C for 30 min and stained with 0.01% (w/v) crystal violet solution at room temperature for 10 min. The number of colonies (defined as >50 cells) visible to the naked eye was counted directly on the plate. Each experiment consisted of two replicates and at least three individual experiments were performed. Statistical significance was calculated from three independent experiments.