Apoptosis assay

HCC cells (3×10⁵ cells per well) were seeded into a 6-well plate for 24 h. The cells were treated with a series of cisplatin concentrations (1,000-5,000 ng/ml) for 72 h. The cells were harvested and washed with PBS twice for 5 min per wash. The cells were suspended and incubated with propidium iodide (PI)-containing Annexin-V-FLUOS labeling solution (Roche Diagnostics) for 15 min at room temperature. Stained cells were analyzed with a flow cytometer (BD Biosciences). For each experiment, 10,000 gated events were acquired to analyze the fluorescence intensity for Annexin-V (excitation: 488 nm, emission: 518 nm) and PI (excitation: 488-540 nm, emission: 617 nm). Early apoptotic cells were defined as Annexin V-positive and PI-negative cells. Late apoptotic cells were defined as Annexin V-positive and PI-positive cells. The total number of apoptotic cells included early and late apoptotic cells (18). Each experiment was analyzed in triplicate and at least three independent experiments were performed.