Inhibition of ABCG2 decreases SP cells, but not sphere-forming ability.

(A) SP analysis of PANC-1 cells. PANC-1 W/T cells, clone #2 cells, and clone #4 cells were stained with Hoechst 33342 and analyzed by flow cytometry. Verapamil was used as a negative control. Percentages indicate the rates of SP cells. (B) Sphere-forming ability of PANC-1 cells. PANC-1 W/T cells, clone #2 cells, and clone #4 cells were seeded in 96-well ultra-low attachment plates at 1, 10, 100, and 1000 cells/well. At 1 week later, sphere-forming wells were counted, and CSC frequency was calculated using the ELDA website. The chi-square test was performed to determine statistically significant differences. (C and D) Tumorigenicity of PANC-1 clone #2 and #4 cells. Growth curves of tumors derived from PANC-1 clone #2 and clone #4 cells. 1000 cells (C) and 10000 cells (D) were injected into BALB/c-nu/nu mice, and tumors were measured weekly. Data are shown as means±SD. Asterisks indicates a statistically difference (P(E) SP analysis of ABCG2 knockdown PANC-1 clone #2 cells. PANC-1 clone #2 cells were transfected with ABCG2 siRNAs. At 2 days after transfection, the cells were analyzed by an SP assay. Verapamil was used as a negative control. Percentages indicate the rates of SP cells. (F) Sphere-forming ability of ABCG2 knockdown PANC-1 clone #2 cells. PANC-1 clone #2 cells were transfected with ABCG2 siRNAs. At 1 day after transfection, the cells were seeded in 96-well ultra-low attachment plates at 1, 10, 100, and 1000 cells/well. At 1 week later, sphere-forming wells were counted, and CSC frequency was calculated using the ELDA website. The chi-square test was performed to determine statistically significant differences. N.S.: No significant difference. (G) SP analysis of Ko143-treated PANC-1 clone #2 cells. PANC-1 clone #2 cells were treated with Ko143 at a concentration of 0, 10, or 20 μM for 24 h and analyzed by an SP assay. Verapamil was used as a negative control. Percentages indicate the rates of SP cells. (H) Sphere-forming ability of Ko143-treated PANC-1 clone #2 cells. PANC-1 clone #2 cells were seeded in 96-well ultra-low attachment plates at 1, 10, 100, and 1000 cells/well with Ko143 at a concentration of 0, 10, or 20 μM. At 1 week later, sphere-forming wells were counted, and CSC frequency was calculated using the ELDA website. The chi-square test was performed to determine statistically significant differences. N.S.: No significant difference.