Side population (SP) analysis and emodin treatment. (A) Chemical structure of emodin. (B) The proportion of SP cells in human gallbladder carcinoma cell line SGC-996 was 1.0% (B-a), which reduced to 0% in the presence of verapamil (B-b). For checking the direct effect of emodin on Hoechst dye exclusion assay, cells were given at the concentrations of 20 μM (B-c), 40 μM (B-d), and 60 μM (B-e) emodin together incubating for 24 h and the effect on SP was analyzed by flow cytometry. (C) Similar to (B), the proportion of SP cells in GBC-SD was 0.8% (C-a) and, as a control, verapamil was added to inhibit the efflux of Hoechst 33342 (C-b). SP cells were reanalyzed after 20 μM (C-c), 40 μM (C-d), and 60 μM (C-e) emodin treatment, respectively. (D) Under experimental conditions with treatment of emodin, a gradual decrease of SP was seen in the SP fraction dose dependently. The ratio of SP cells was calculated: SP/Total cells. Each experiment was repeated 3 times.