Overexpression of Id genes in BMMs inhibits osteoclastogenesis. (A-B) BMMs were transduced with pMX-IRES-EGFP (control), Id1, Id2, or Id3 retrovirus and cultured for 3 days with M-CSF alone or M-CSF and TRANCE. (A) Cultured cells were fixed and stained for TRAP (original magnification, × 100). (B) Numbers of TRAP-positive multinucleated cells were counted. (C-D) BMMs were transduced with control (pMX-IRES-EGFP), Id1, Id2, or Id3 retrovirus and cultured for 3 days with M-CSF and various concentrations of TRANCE as indicated. (C) TRAP activity was determined by measuring OD values at 405 nm. (D) MTT assay results were determined by measuring OD values at A570. (E-F) BMMs were transduced with pMX-IRES-EGFP (control), Id1, Id2, or Id3 retrovirus and cocultured for 6 days with osteoblasts in the presence of 1,25(OH)₂D₃ and PGE₂. (E) Cultured cells were fixed and stained for TRAP (original magnification, × 100). (F) Numbers of TRAP-positive multinucleated cells were counted (*P < .05). Results are representative of at least 3 independent sets of similar experiments (A-F). Data represent means ± SDs of triplicate experiments. Cells were observed under a Leica DMIRB microscope equipped with an N Plan 10×/0.25 objective lens (Leica, Wetzlar, Germany). Images were obtained through a Leica IM50 using Leica IM 4.0 software (Leica, Cambridge, United Kingdom).