Figure 2.

Ablation of Shn3 in osteoblasts enhances osteogenesis and angiogenesis in vivo. (a) Representative μCT images of the trabecular bone in the distal femur (left) and bone volume/total volume (BV/TV) (right) in Shn3^f/f (n = 7) and Shn3^dmp1 (n = 8) male mice at 8-weeks of age. Scale bars, 1mm. (b) Representative histomorphometric images of the L3 vertebrae (left) and BV/TV (right) in Shn3^f/f (n = 6) and Shn3^dmp1 (n = 4) male mice at 8-weeks of age. Scale bars, 500μm. (c, d) Representative images of calcein double labeling (c) and quantification of histomorphometric parameters (d) of the L3 vertebrae in Shn3^f/f and Shn3^dmp1 male mice at 8-week of age. Trabecular mineral apposition rate (MAR, mm day⁻¹), bone formation rate/bone surface (BFR/BS, mm³ mm⁻² yr⁻¹), osteoclast number/bone perimeter (No. Oc./B. Pm) and osteoblast surface/bone surface (Ob.S/BS, %). Scale bars, 300μm. MAR and BFR/BS: n = 6 per group; No. Oc./B. Pm and Ob.S/BS: n = 5 per group. (e) Representative confocal images (n = 3 total images per group) of femur sections from 2-week old Shn3^f/f and Shn3^dmp1 male mice with EMCN (red) and CD31 (green). Growth plate is marked with a dashed line. Scale bars, 100 μm. (f, g) Representative flow cytometry plots (f) and relative frequency of CD31^hiEMCN^hi endothelial cells (g) from the femurs of 2-week-old male Shn3^dmp1 (n = 6) and Shn3^f/f (n = 5) mice. (h, i) Representative μCT images of the trabecular bone in the distal femur metaphysis (h) and relative quantitative analysis of BV/TV (i) in Shn3^f/f (oil =7; tamoxifen =7) and Shn3^ocn-ert2 male mice (oil =6; tamoxifen =8). Analysis was performed 12-weeks after tamoxifen injection into 4-week old mice. Scale bars, 1mm. (j) Representative confocal images (n = 3 total images per group) of EMCN (red) and DAPI (blue) immunostained femur sections from Shn3^f/f and Shn3^ocn-ert2 male mice at 4-weeks after tamoxifen injection into 4-week old mice. Growth plate is marked with a dashed line. Scale bars, 300 μm. (k, l) Representative flow cytometry plots (k) and relative frequency of CD31^hiEMCN^hi endothelial cells (l) from the femurs of Shn3^f/f and Shn3^ocn-ert2 male mice (n = 5 per group), 6-weeks after tamoxifen injection into 4-weeks old mice. Values represent mean ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001 all by an unpaired two-tailed Student’s t-test or by one-way ANOVA followed by a Dunnett’s test in all panels.