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. 2009 Sep 21;119(10):3024–3034. doi: 10.1172/JCI38716

Figure 1. TNF-induced expression of NF-κB p100 inhibits osteoclastogenesis.

Figure 1

(A) WT mouse OCPs, cultured from splenocytes with M-CSF for 3 days, were treated with RANKL or TNF for the indicated times. NF-κB proteins in whole-cell lysates were determined by Western blot. Experiments were repeated at least twice with similar results. P, PBS; R, RANKL 10 ng/ml; T, TNF 20 ng/ml. (B) WT or Nfkb2–/– OCPs were treated with RANKL or TNF directly on plastic or bone slices in 96-well plates in the presence of M-CSF for 2 and 5 days, respectively, to induce osteoclasts (OCs) and resorption pits. Top: Representative TRAP-stained osteoclasts (original magnification, ×4) and toluidine blue–stained pits (original magnification, ×20). Bottom: Osteoclast number and resorption pit area (n = 4/group; *P < 0.05 vs RANKL). (C) Nfkb2–/– or WT OCPs were infected with GFP, p100, or p52 retroviruses for 2 days and treated with TNF for 2 more days. Osteoclast numbers were counted (left panel; *P < 0.05 versus GFP), and the infection efficiency was confirmed by Western blot from the infected WT OCPs (right panels). (D) Murine TNF (0.5 μg in 10 μl PBS) or 10 μl PBS were injected twice daily over the calvariae of 4-week-old Nfkb2–/– or Nfkb2+/– control mice for 5 days (n = 4/group). The number of osteoclasts/mm bone surface, percentage of osteoclast surface/bone surface, and percentage of eroded surface/bone surface were measured in TRAP-stained calvarial bone sections, and serum TRAP5b was tested with ELISA.