Figure 3

PDGF-D induces osteoclast differentiation in vitro. (a) RT–PCR analysis of α-PDGFR and β-PDGFR mRNA in RAW264.7 and NIH 3T3 cells. (b) Full-length rPDGF-D was incubated with CM collected from RAW264.7 cells (left panel) or with serum-free medium (right panel). At indicated time points, the processing of rPDGF-D was assessed by immunoblot analysis in a reducing condition using anti-PDGF-D antibody (8D2). (c) TRAP staining of RAW264.7 cells treated with 12.5 ng/ml (0.5 nM) rPDGF-B, 30 ng/ml (1.5 nM) rRANKL or 40 ng/ml (0.5 nM) rPDGF-D for 6 days. Note that RANKL exists as a monomer and yet works as a trimer while binding with RANK on cell surface. Pictures were taken under × 400 magnification. (d) RAW264.7 cells were treated with indicated concentration of PDGF-B, PDGF-D and RANKL for 6 days. An average number of differentiated osteoclast cells defined as TRAP-positive multinucleated cells (more than three nuclei/cell) in five low-power fields per treatment is shown. ^*P<0.01. (e) RAW264.7 cells were treated with 12.5 ng/ml (0.5 nM) PDGF-B, 40 ng/ml (0.5 nM) PDGF-D and 30 ng/ml (1.5 nM) RANKL for indicated days. The fold induction of TRAP mRNA normalized to the GAPDH level at indicated time points is graphed. TRAP mRNA level in control group (containing 0.5% FBS) was given as 1. Statistical analyses were based on three independent experiments performed in triplicates. P values were labeled between groups. (f) NIH3T3 cells were treated with 0.5 nM rPDGF-B (12.5 ng/ml) or rPDGF-D (40 ng/ml, activated by matriptase) for 10 min. The levels of p-β-PDGFR, β-PDGFR, pERKs and ERKs were analyzed by immunoblot analyses. (g) RAW264.7 cells were treated with concentrated CM from LNCaP-neo and LNCaP-PDGF-D cells for 6 days followed by TRAP staining. An average number of differentiated osteoclast cells by each treatment were graphed. ^*P<0.01.