Effects of Notch ligand blockade and Notch receptor stimulation on osteoclastogenesis from human PBmono. (a) Osteoclasts were differentiated from human PBmono as described in Figure 3. DAPT (10 μM), DMSO as a control, 20 μg/ml of the indicated mAb (hD1, MHD1-314; hD4, MHD4-46; hJ1, MHJ1-152; hJ2, MHJ2-473), or control mouse IgG (mIg) was added on Day 0 and Day 3. (b) Osteoclasts were induced as described in (a) and mRNA levels of Hes1 and beta-actin were determined by RT-PCR. (c and e) Osteoclast precursors were pre-incubated with Fc(gamma)R-binding inhibitor and then differentiated on the culture plate immobilized with 10 μg/ml of the indicated Fc chimera or control human IgG-Fc fragment (hIgG-Fc) (c), or with 5 μg/ml of the indicated mAb (hN1, MHN1-128; hN2, MHN2-25; hN3, MHN3-21; hN4, MHN4-2) or mIg (e). (d) Osteoclasts were differentiated as described in (a) or (c) in the presence of a low dose of rhRANKL (12.5 ng/ml). (a, c-e) Osteoclasts were stained for TRAP. Representative stainings are shown (a, c and e). Original magnification: ×100. Data are indicated as the mean TRAP-positive MNCs per culture well ± SD of triplicated wells..#, P < 0.001; versus DMSO control. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus mIg or hIgG-Fc control. Similar results were obtained in three independent experiments.