Figure 2.

Modulation of the EMT and metastatic properties of PC3 cells following overexpression of RANKL. (A) A cell migration assay was performed to compare wound healing in PC3^Wild and PC3^RANKL+ cells. PC3^RANKL+ cells showed a significant increase in migratory capacity, as evident from the decreased wound gap 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 PC3^Wild and PC3^RANKL+ cells. PC3^RANKL+ cells showed a significant increase in invasiveness. 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 PC3^RANKL+ cells. β-Actin was used as a loading control. Significant differences were observed at *p < 0.05, compared with the control. (D) Expression of E-cadherin, Vimentin, β-catenin, MMP-9, IL-6 and β-actin in PC3^Wild and PC3^RANKL+ cells, as measured by western blotting. β-Actin was used as a loading control. (E) Immunoprecipitation for β-catenin and TCF-4 in PC3^Wild and PC3^RANKL+ cells. Each blot was obtained under the same experimental conditions and the data are representative of three separate experiments with comparable results. (F) TOP/FOP luciferase reporter assays in PC3^Wild and PC3^RANKL+ cells. Significant differences were observed at *p < 0.05, compared with the control.