Figure 1.

EMT and metastatic properties of PC3 cells following hRANKL treatment. (A) Treatment with hRANKL led to a significant increase in the migratory capacity of PC3 cells, as evident from the decrease in the wound gap distance at 48 h. Magnification, × 100; scale bar, 100 μm. The data in the associated graphs are expressed as the mean ± SD. (B) Transwell invasion assays were performed to compare the invasiveness of hRANKL-treated PC3 cells and untreated cells. Treatment with hRANKL led to a significant reduction in the invasiveness of PC3 cells. Magnification, × 100; scale bar, 100 μm. Data in the associated graphs are expressed as the mean ± SD. (C) Real-time quantitative polymerase chain reaction for the analysis of EMT and metastasis markers in hRANKL-treated PC3 cells. β-Actin was used as a loading control. Significant differences were observed at *p < 0.05, compared with the control. (D) Expression of EMT and metastasis-related proteins E-cadherin, Vimentin, β-catenin, MMP-9, IL-6, c-MYC, TCF-4 and β-actin in hRANKL-treated PC3 cells was measured by western blotting. β-actin was used as a loading control. (E) Western blot analysis of MAPK phosphorylation in PC3 cells incubated with 1 μg/mL hRANKL for 0, 5, 15 and 30 min. (F) Co-immunoprecipitation of β-catenin and TCF-4 in hRANKL-treated PC3 cells. Results are representative of three separate experiments with comparable results. (G) TOP/FOP luciferase reporter assays in PC3^Wild and PC3^RANKL+ cells. Significant differences were observed at *p < 0.05, compared with the control.