RBP-J deficiency allows TNF to induce osteoclastogenesis and bone resorption independent of RANK signaling. (A) Quantitation of TRAP-positive MNCs of the cell cultures from Rbpj^+/+ and Rbpj^ΔM/ΔM BMMs treated with RANKL, RANKL together with OPG (100 ng/ml), or RANK-Fc (5 µg/ml) in the presence of M-CSF for 4 d. (B) Quantitation of TRAP-positive MNCs of the cell cultures from Rbpj^+/+ and Rbpj^ΔM/ΔM BMMs treated with TNF, TNF together with OPG (100 ng/ml), or RANK-Fc (5 µg/ml) in the presence of M-CSF for 4 d. TRAP staining (C) and quantitation (D) of TRAP-positive MNCs in 5-d osteoclastogenic cell cultures from Rank^−/− and Rank^−/−Rbpj^ΔM/ΔM BMMs. Bar, 100 µm. **, P < 0.01. (E and F) TRAP staining of mouse whole calvaria (E) and of calvarial histological sections (F) obtained from 4-wk-old Rank^−/− and Rank^−/−Rbpj^ΔM/ΔM mice injected with PBS or TNF (150 µg/kg body weight). n = 5 per group. Bars: 0.5 cm (E); 100 µm (F). (G) Histomorphometric analysis of calvaria from Rank^−/− and Rank^−/−Rbpj^ΔM/ΔM mice. N.Oc/BS, number of osteoclasts per bone surface; Oc.S/BS, osteoclast surface per bone surface. n = 5 per group. **, P < 0.01. (H) The concentration of serum TRAP obtained from 4-wk-old Rank^−/− and Rank^−/−Rbpj^ΔM/ΔM mice injected with PBS or TNF. n = 5 for Rank^−/− mice, and n = 6 for Rank^−/−Rbpj^ΔM/ΔM mice. **, P < 0.01. Data are representative of at least three (A–D) or two (E–H) independent experiments.