(A-D) 12 wk-old female and male mice were injected with pI-pC and sacrificed 5 wk later.
(A) mRNA levels of FoxOs in calvaria (calv), vertebra (vert), liver or spleen from male mice determined by qRT-PCR (n=8 mice/group).
(B) BMDs determined by DEXA 1 d before and 5 wk following pI-pC injections (n=10-17 mice/group). *p<0.05 vs respective Mx-Cre^- by Student's t-test
(C) Cortical width measured by micro-CT in femurs (n=10-17 mice/group). *p<0.05 vs respective Mx-Cre^- by Student's t-test
(D) Histomorphometric analysis of longitudinal undecalcified sections of L1-L3 vertebrae of male mice (n=6-7 mice/group). BA/TA=bone area per tissue area; Tb=trabecular.
*p<0.05 by Student's t-test.

(A-C) Measurements were made using bone from the animals described in Figure 2.
(A) Histomorphometric analysis of longitudinal undecalcified sections of L1-L3 vertebrae of male mice (n=6-7 mice/group). Ob=osteoblast; Oc=osteoclast.
(B) p66^shc phosphorylation determined by Western blot in vertebral lysates; each lane represents one animal. The mean ratio ± SD of phosphorylated p66^shc to β-actin is depicted numerically at the bottom of the corresponding blots.
(C) Osteoblasts and osteocyte apoptosis in sections of the same mice as in A. In the photomicrographs apoptotic osteocytes and osteoblasts (stained brown by ISEL, original magnification ×630) are indicated by the arrows (n=7-8 mice/group).
*p<0.05 by Student's t-test.

(A-D) Cells were obtained from the mice described in figure 2.
(A) mRNA levels of FoxOs by qRT-PCR and protein levels of FoxOs by Western Blot in calvaria-derived osteoblastic cells cultured for 8 d. *p<0.05 by Student's t-test.
(B) Caspase 3 activity in cells described in A, cultured in the presence of vehicle or NAC (1 mM) for 24 h. *p<0.05 vs veh in Mx-Cre^-, # p<0.05 vs veh in Mx-Cre⁺ by ANOVA.
(C) p66^shc phosphorylation determined by western blot in lysates from calvaria cells described in B. The ratio of phosphorylated p66^shc to β-actin is depicted numerically at the bottom of the corresponding blots.
(D) mRNA levels of the indicated genes in bone marrow-derived stromal cells cultured for 8 d.
*p<0.05 by Student's t-test
(E) Bone marrow-derived stromal cells were treated with vehicle or rosiglitazone (RGL; 5 nM/ml) for 11 days. Lipid was visualized with Oil Red O and quantified as described in Methods. *p<0.05 vs RGL in Mx-Cre^-, †p<0.05 vs vehicle in Mx-Cre^- by ANOVA.

(A-D) Cells were obtained from the mice described in figure 2.
(A) mRNA levels of FoxOs by qRT-PCR and protein levels of FoxOs by Western Blot in mature osteoclasts generated from non-adherent bone marrow cells cultured with M-CSF and RANKL for 5 d; ND=not detected.
(B) Caspase 3 activity and (C) mRNA levels of the indicated genes in cells described in A. CtR=calcitonin receptor; CtsK=cathepsin K.
(D) Number of TRAP positive cells generated from bone marrow cells cultured with M-CSF and RANKL for 6 d. TRAP=tartrate-resistant acid phosphatase.
*p<0.05 by Student's t-test.

(A) Mice harboring a transgene consisting of a transcriptional/translational stop cassette, flanked by loxP sites, inserted between the broadly-expressed hybrid CMV-chicken β-actin promoter and a cDNA encoding an HA-tagged human FoxO3 WT, designated FoxO3^C mice were crossed with mice expressing the Cre recombinase under the control of the OCN promoter. The loxP-flanked stop cassette is removed only in cells expressing Cre resulting in FoxO3 expression in that cell type. PA=polyadenilation site.
(B) mRNA levels of the FoxO3 transgene in calvaria, vertebra (L5), liver and spleen of 4 wk-old mice determined by qRT-PCR (n=3 mice/group); ND=not detected.
(C) mRNA levels of the indicated FoxO-target genes in calvaria from 4 wk-old mice (n=3 mice/group).
(D) Calvaria-derived osteoblastic cells isolated from newborn FoxO3^C and WT littermates infected with Ad-Cre for FoxO3 overexpression and transfected with a FoxO-luc reporter construct. Luc activity was measured 24 h later.
*p<0.05 by Student's t-test.

(A) Spine BMD by DEXA and (B-C) micro-CT measurements in the vertebra (L5) of 16 wk-old mice (n=7-14 mice/group). BV/TV=bone volume per total volume; Tb=trabecular. Representative images of vertebral cancellous bone are shown. *p<0.05 vs respective OCN-Cre by Student's t-test
(D) Histomorphometric analysis of longitudinal undecalcified sections of L1-L3 vertebrae of 16 wk-old male mice (n=6 mice/group). *p<0.05 by Student's t-test. BA/TA=bone area per tissue area.

(A-C) Measurements were made using bones from the animals described in Figure 6.
(A) Histomorphometric analysis of longitudinal undecalcified sections of L1-L3 vertebrae of male mice (n=7 mice/group). Ob=osteoblasts; BFR=bone formation rate.
(B) p66^shc phosphorylation determined by Western blot in vertebral lysates; each lane represents one animal. The mean ratio ± SD of phosphorylated p66^shc to β-actin is depicted numerically at the bottom of the corresponding blots.
(C) Osteoblasts and osteocyte apoptosis in sections of the same mice as in A.
(D) mRNA levels of FoxO3 transgene by qRT-PCR and protein levels of HA-FoxO3 and Foxo3 by Western blot in bone marrow-derived osteoblastic cells cultured for 18 d with ascorbic acid.
(E) Caspase 3 activity in cells described in D, cultured in the presence of vehicle or H₂O₂ at the indicated concentrations for 16 h. *p<0.05 vs no H₂O₂ by ANOVA. ^#p<0.05 vs respective H₂O₂ dose in OCN-Cre by Student's t-test.
(F) Left panel: number of TRAP positive cells generated from bone marrow cells cultured with M-CSF and RANKL for 6 d. Right panel: osteoclast number was determined in the same sections as in A. TRAP=tartrate-resistant acid phosphatase; Oc=osteoclast.
*p<0.05 by Student's t-test.