Figure 5

CXCL10-neutralizing antibody rescues enhanced osteoclastogenesis. (a–c) CXCL10-neutralizing antibody (20 ng/ml) or IgG was added to co-cultures of wild-type BMCs with Men1-deficient primary osteocytes isolated from Men1^{gtRosaCreERT2} mice and treated with 4-OHT. Co-cultures were stained for TRAP activity (a). Number of multinucleated TRAP-positive cells (b) and their area (c) were determined (n=3). (d–f) CXCL10-neutralizing antibody (20 ng/ml) or IgG was added to co-cultures of wild-type BMCs with primary osteocytes isolated from Men1^flox and Men1^{gtRosaCreERT2} mice and treated with 4-OHT. The co-cultures were seeded on bone slices. Resorption pits were stained with toluidine blue (d). Pit area (e) and CTX levels in the supernatant (f) were determined (n=3). (g–j) Organ cultures of calvaria from neonatal Men1^Dmp1Cre mice were treated with IgG or CXCL10-neutralizing antibody. TRAP staining was performed and osteoclasts were visualized (arrows) (g). Acp5 (h) and Ctsk (i) mRNA levels were determined by QRT-PCR. CTX levels in the supernatant (j) were determined by ELISA (n=3 or 4). (k–m) IgG or CXCL10-neutralizing antibody was subcutaneously injected over the calvaria of 12-week-old female Men1^flox and Men1^Dmp1Cre mice. TRAP staining was performed (k). Acp5 (l) and Tnfrsf11a (m) mRNA levels were determined by QRT-PCR (n=4). *P<0.05, **P<0.01, ***P<0.001. Data are represented as mean±S.E.M. Scale bar: 25 μm