Figure 3: The effects of genetic ablation of Ckip-1 within osteoblast on bone formation and Smad-dependent BMP signaling in glucocorticoid-treated mice.

(a) Schematic diagram of the experimental design. (b) Representative in vivo micro-CT images of the time-course changes in three-dimensional trabecular architecture of the left proximal tibiae from each group. Scale bar: 100 um. (c) The time-course changes in micro-CT parameter (BMD) of the left proximal tibia from each group. (d) Representative micrographs of newly mineralized bone assessed by xylenol (red) and calcein (green) labeling at the left proximal tibiae from each group after 4 weeks. Scale bar: 50 μm. (e) The changes in the bone histomorphometric parameter (MAR and Ob.S/BS) of the left proximal tibiae from each group after 4 weeks. (f) The representative electrophoretic bands of the time-course changes in the ubiquitination of total Smad1 in bone tissue level from each group. The mice were treated with MG132 (2 mg/kg) through intraperitoneal injection 24 hours before sample collections. Polyubiquitinated Smad1 was detected by anti-ubiquitin immunoblot analysis after precipitation of Smad1 in the cell lysis of MC3T3-E1 cells pretreated with proteasome inhibitor MG132. (g) The representative immunofluorescence images for the time-course changes in the co-expression of p-Smad1/5 (red) and OCN + (green) cells of the right proximal tibiae from each group. Merged images with DAPI staining showed cells co-staining of p-Smad1/5 with Osteocalcin (arrow indicated). Scale bar: 25 μm. Note: The data were mean ± s.d. *P < 0.05 for WT-PNL group vs cKO-PNL group. WT: wildtype littermate mice; cKO: osteoblast-specific Ckip-1 knockout mice. Base: Baseline of before glucocorticoid treatment. PNL: prednisolone treatment; VH: Vehicle treatment.