Analysis of expression of RUNX2 and RANKL in PC3 cells. A and B. RT-PCR and immunoblotting analysis of expression of RUNX2 in PC3 (lanes 1), HPR1 (lane 2) and BPH (lane 3) cells is shown. C-E: The effects of SiRNA to RUNX2 on RUNX2 (C) and RANKL (D) protein levels in total cellular lysates (C and D) and conditioned medium (E). Immunoblotting analysis in conditioned medium represents the secreted levels of RANKL. Untransfected (−) or scrambled SiRNA (Sc) transfected PC3 cells were used as controls (B-E). GAPDH was used as a loading control for RT-PCR (A) and Western blot (B -D) analyses. The loading control for the conditioned medium is shown by the use of Coomassie blue staining of the blot (F). G - I: Immunostaining and confocal microscopy analysis of distribution of RUNX2 (red; H) and RANKL (green; I) in PC3 cells. Distribution of both RANKL (red) and RUNX2 (green) are shown in panel G. Results shown are representative of three independent experiments. Scale bar: 50 μm.