Figure 3

Expression of Notch receptors and ligands during osteoclastogenesis from human PBmono. Reactivity of anti-human Notch receptor mAbs (a) or anti-human Notch ligand mAbs (b) against CHO transfectants. Open histograms indicate the staining with anti-Notch1 (MHN1-128), anti-Notch2 (MHN2-25), anti-Notch3 (MHN3-21), anti-Notch4 (MHN4-2), anti-Dll1 (MHD1-314), anti-Dll4 (MHD4-46), anti-Jagged1 (MHJ1-152) or anti-Jagged2 (MHJ2-473) mAb. Filled histograms indicate the staining with control mouse IgG (a-c). (c) PBmono cultured with rhM-CSF for 72 hours were used as osteoclast precursors (Day 0). These cells were further cultured with rhM-CSF and rhRANKL to induce osteoclasts. CD14⁺ cells were analyzed by flow cytometry on days 0, 3 and 5. CD51/CD61⁺CD14⁺ osteoclasts were analyzed on Day 5. For comparison, PBmono were cultured with rhM-CSF alone for eight days and CD14⁺ cells were analyzed as macrophages (Mø), or with rhGM-CSF and rhIL-4 for seven days with LPS stimulation for the last 48 hours and CD11c⁺ cells were analyzed as dendritic cells (DC). Open histograms indicate the staining with MHN1-128 (Notch1), MHN2-25 (Notch2), MHN3-21 (Notch3), MHN4-2 (Notch4), MHD1-314 (Dll1), MHD4-46 (Dll4), MHJ1-152 (Jagged1), or MHJ2-473 (Jagged2). (d) Osteoclasts were induced as described in (c). Messenger RNA levels of Notch ligands were measured by RT-PCR on days 0, 3 and 5.