Fig. 5

The knockout of Numb and Numbl in osteoblasts boosted osteoclastogenesis. a TRAP staining on paraffin sections of WT and DKO femurs to show osteoclasts on the bone surface. Scale bar, 50 μm. b The concentration of tartrate-resistant acid phosphatase 5 (ACP5, n = 12) in the serum of WT or DKO mice. Data represent the mean ± SEM, *P < 0.05. Histomorphometry based on TRAP staining: (c) the number of osteoclasts per trabecular bone perimeter (N.Oc/T.Pm, n = 10); d the osteoclast surface rate (Oc.S/BS, n = 10); (e) the average osteoclast surface (Oc surface, n = 10). Data shown represent the mean ± SEM, *P < 0.05. f Co-culture of WT or DKO calvaria (bottom) osteoblasts with BMC (top) from WT or DKO mice in 48-well plates, TRAP-stained and haematoxylin counterstained. g The number of osteoclasts were counted and are shown as the mean ± SEM, *P < 0.05. h Representative images of bone resorption pits from a scanning electron microscope (SEM). Scale bar, 400 μm. i RAW264.7 cells were co-cultured on the surface of ΔN/NL or CTRL osteoblasts in 48-well plates and were TRAP-stained. j Numb overexpression in ΔN/NL osteoblasts enhanced osteoclastogenesis. ΔN/NL osteoblasts were transfected with a GFP-m-Numb plasmid. Two days after transfection, RAW264.7 cells were seeded on the surface of osteoblasts, and the wells were stained with TRAP 10 days later. k Transwell cultures of ΔN/NL or CTRL osteoblasts on small transwell inserts with 0.4 μm pores; RAW264.7 cells were co-cultured in lower 24-well plates, and TRAP staining was performed 10 days later