Fig. 2

Inducible deletion of shn3 in osteoblasts increases bone accrual in adult mice. a–d A single dose of 4 × 10¹¹ genome copies of rAAV9-Egfp was intravenously (i.v.) injected into 2-month-old male mice and EGFP expression in individual tissues was monitored by IVIS-100 optical imaging 2 weeks post injection. y-axis, radiant efficiency (p/s/cm²/sr/μW/cm²) (a). EGFP expression was assessed by fluorescence microscopy on cryo-sectioned heart, liver (b), and femur (c). A high-magnification image of EGFP-expressing osteoblasts in the femur (c, right). Immunoblot of tissue lysates with an anti-GFP antibody (d). e–g Two-month-old female Ocn-Ert;Rosa^mT/mG and Shn3^Ocn-Ert;Rosa^mT/mG mice were treated with 100 mg/kg of tamoxifen for 5 consecutive days; 2 months later, femurs were cryo sectioned to identify EGFP-expressing osteoblasts (e). Femoral trabecular bone mass was assessed by microCT. Representative 3D reconstruction (f) and relative quantification (g) are displayed. Trabecular bone volume/total volume (Tb. BV/TV), trabecular thickness (Tb.Th), trabecular number per cubic millimeter (Tb.N), and trabecular space (Tb. Sp) (n = 6 per group). h–k A single dose of 4 × 10¹¹ genome copies of rAAV9-Egfp or rAAV9-Cre was i.v. injected into 3-month-old male Shn3^fl/fl;Rosa^mTmG mice. After 2 months, cre and shn3 mRNA levels were assessed in tibial bone RNA and normalized to hprt (h). Fluorescence microscopy was performed on cryo-sectioned femurs to identify EGFP-expressing cells (i) and femoral trabecular bone mass was assessed by microCT. Representative 3D reconstruction (j) and relative quantification (k) are displayed. Trabecular bone volume/total volume (Tb. BV/TV), trabecular thickness (Tb. Th), trabecular number per cubic millimeter (Tb. N), and connective density (Conn. Dn) (n = 6 per group). Scale bars: b 50 μm; c 500 μm (left), 75 μm (right); e 25 μm; f, j 1 mm; i 250 μm. Values represent mean ± SD: *P < 0.05; **P < 0.01; and ***P < 0.001 by an unpaired two-tailed Student’s t-test (g, h, k)