Journal: Arthritis Research & Therapy

Article Title: Differential regulation of osteoclastogenesis by Notch2/Delta-like 1 and Notch1/Jagged1 axes

doi: 10.1186/ar3758

Figure Lengend Snippet: 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.

Article Snippet: The human IgG Fc fragment was purchased from Acris Antibodies (Herford, Germany).

Techniques: Reverse Transcription Polymerase Chain Reaction, Incubation, Blocking Assay, Knock-Out, Staining

Journal: Arthritis Research & Therapy

Article Title: Differential regulation of osteoclastogenesis by Notch2/Delta-like 1 and Notch1/Jagged1 axes

doi: 10.1186/ar3758

Figure Lengend Snippet: 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.

Article Snippet: The human IgG Fc fragment was purchased from Acris Antibodies (Herford, Germany).

Techniques: Expressing, Staining, Cell Culture, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction

Journal: Arthritis Research & Therapy

Article Title: Differential regulation of osteoclastogenesis by Notch2/Delta-like 1 and Notch1/Jagged1 axes

doi: 10.1186/ar3758

Figure Lengend Snippet: 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.

Article Snippet: The human IgG Fc fragment was purchased from Acris Antibodies (Herford, Germany).

Techniques: Reverse Transcription Polymerase Chain Reaction, Incubation, Binding Assay, Staining