Effects of Notch ligand blockade and Notch receptor stimulation on osteoclastogenesis from mouse BM. (a) Osteoclasts were induced from mouse BM as described in Figure 1. DAPT (10 μM), DMSO as a control, 20 μg/ml of the indicated mAb (D1, HMD1-5; D4, HMD4-2; J1, HMJ1-29; J2, HMJ2-1), or control hamster IgG (Ham) was added on Day 0 and Day 3. (b) Osteoclasts were induced as described in (a) and mRNA levels of Hes-1 and beta-actin were determined by RT-PCR on Day 3. (c and e) Osteoclast precursors were pre-incubated with Fc Block and then differentiated on the 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 (N1, HMN1-12; N2, HMN2-29; N3, HMN3-133; N4, HMN4-14) or Ham (e). (d) BM cells from Dll1-conditional knockout or littermate control mice were differentiated into osteoclasts. ***, P < 0.001. (a, c-e) Osteoclasts were stained for TRAP. Representative stainings are shown. 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 Ham or hIgG-Fc control. Similar results were obtained in three independent experiments.