Defective maturation of CD45KO osteoclasts in vitro involving impaired expression of MMPs, DC-STAMP, and Src kinase. (A) BM-derived osteoclasts from WT (top) and CD45KO (bottom) mice immunolabeled for CD45 expression (green), and stained for polymerized actin (red) and nuclear DNA (blue). Bars, 20 μm. (B) TRAP staining (purple) of BM-derived WT (top) and CD45KO (bottom) osteoclasts. Bars, 200 μm. (C and D) Semiquantitative PCR analysis for DC-STAMP mRNA levels in WT and CD45KO BM-derived cells. (C) Representative PCR image. (D) Summary of four independent experiments showing the ratio between DC-STAMP and GAPDH mRNA expression (±SE; *, P < 0.01). (E) Conditioned medium of WT versus CD45KO BM-derived osteoclasts was tested for the activity of secreted MMP-9 in a gelatin zymography assay. (F) WT and CD45KO BM-derived osteoclasts cultured with or without G-CSF were immunolabeled for expression of MT1-MMP (green), and stained for polymerized actin (red) and nuclear DNA (blue). Bar, 10 μm. (G) TRAP staining (purple) of BM-derived WT (top) and CD45KO (bottom) osteoclasts cultured in vitro in the presence of DMSO vehicle (left) or the Src inhibitor PP2 (right). Bars, 200 μm. (H) Src activity assay for osteoclast precursors incubated with 1 μM PP2 or DMSO (ctrl) for 5 d. Values indicate the fold changes of WT control mice ± SE (indicated by a horizontal line), showing a representative experiment.