Curcumin induces apoptosis in GBC-SD cells. (A) Cells were incubated with curcumin (3.75, 7.5, and 15 μmol/L) for 48 h, followed by staining with annexin-V/PI. The Q3 quadrant (annexin V−/PI−), Q4 quadrant (annexin V+/PI−) and Q2 quadrant (annexin V+/PI+) indicate the percentage of normal cells, early apoptosis and late apoptosis, respectively. (B) Apoptotic nuclear morphology changes induced by curcumin (3.75, 7.5, and 15 μmol/L) treatment for 48 h, were observed by Hoechst 33342 staining in GBC-SD cell lines. (C) Analysis of the mitochondrial membrane potential (ΔΨm). GBC-SD cells were treated with curcumin (3.75, 7.5, and 15 μmol/L) for 48h and then stained with JC-1. Red fluorescence represents mitochondria with intact membrane potential. Green fluorescence represents de-energized mitochondria. Images were taken with a fluorescence microscope.