Analysis of binding of RUNX2 with RANKL promoter and the effect of RUNX2 knockdown on osteoclast differentiation. A. Chromatin immunoprecipitation (ChIP) assay. ChIP assay was used to determine the RUNX2 binding sites in RANKL promoter. Immunoprecipitates were made with an antibody (rabbit) to RUNX2 (lane 4) or rabbit IgG (lane 3) using lysates made from PC3 cells. DNA from the input (lane 2) and immunoprecipitates (lanes 3 and 4) was analyzed by RT-PCR using primers specific for RUNX2 binding sites on RANKL promoter. As expected, a product size 153 bp was observed in the RT-PCR analysis. The experiment was repeated twice and obtained similar results. B-D: The conditioned media (CM) from PC3 cells untreated (B) or treated with scrambled (C) and SiRNA (D) to RUNX2 were used for osteoclast differentiation in vitro. TRAP-positive osteoclasts are stained in dark purple. Cells were observed under an inverted phase contrast microscope and images were captured (X 200). The results shown are representative of three experiments.