Figure 6

Effect of transfection of integrin-β3 siRNA and cyclic RGDfV peptide on osteoclastogenesis from CD16^- monocytes. (a) CD16^- monocytes transfected with either 1 nM control or integrin-β3 siRNA were cultured in macrophage colony-stimulating factor (M-CSF) (25 ng/ml) + receptor activator of NF-κB ligand (RANKL) (40 ng/ml). Forty-eight hours after the transfection, integrin-β3 mRNA levels were examined by semiquantitative RT-PCR. (b) The expression of the αvβ3 heterodimer was examined by immunostaining and the number of αvβ3-positive cells was counted. (c) Seven days after the transfection of siRNAs, the cells were stained for tartrate-resistant acid phosphatase (TRAP) activity, and the number of TRAP-positive multinucleated cells (MNC) was counted. Results are representative of three to five independent experiments. Data are the mean ± SEM values of duplicate wells. *P < 0.01, control-siRNA vs β3-siRNA. (d) CD16^- monocytes were incubated with M-CSF (25 ng/ml) + RANKL (40 ng/ml) for 2 days, followed by the addition of a medium containing either cyclic RGDfV peptide or dimethyl sulfoxide (DMSO). After incubation for a further 5 days, the number of TRAP-positive multinucleated cells (MNC) was counted. Representative results from three independent experiments are shown. Data are the mean ± standard error of the mean values of triplicate wells. Control, without treatment. **P < 0.03, DMSO vs cyclic RGDfV peptide.