Figure 1. FoxO1 interacts with β-catenin in osteoblasts.

(a) Coimmunoprecipitation (IP) of β-Catenin and FoxO1 in cell lysates from osteoblast cells followed by immunoblot analysis. Blots were representatives of n=3. (b–f) Real-Time PCR analysis of (b) Axin-2, Tcf-1, Tcf-3 and Lef-1 expression in osteoblasts transfected with increasing concentrations of FoxO1 construct. EV denotes Empty vector. Data are representative of 3 independent experiments. ^*p < 0.05 versus EV-transfected cells. (c) Real-Time PCR analysis of Axin-2, Tcf1, Tcf-3 and Lef-1 in the bones of wild type and Ctnnb1^CAosb mice (Identical data are shown in Figure 2b–e). (d) Real-Time PCR analysis of Cyclin D1, Cyclin D2, p27kip1, Sod2 and Gadd45 expression in osteoblasts transfected with increasing concentrations of β-catenin. EV denotes Empty vector. Data are representative of three independent experiments. ^*p < 0.05 versus EV-transfected cells. (e) Quantitative Real-Time PCR analysis of Axin-2, Tcf1, Tcf-3 and Lef-1 in the bones of wild type and FoxO1_osb−/− mice. Total RNA was isolated from flushed long bones. n=4. ^*p < 0.05 versus wild type. (f) Quantitative Real-Time PCR analysis of Cyclin D1, Cyclin D2, p27kip1, Sod2 and Gadd45 gene expression in bones of wild type and Ctnnb1^CAosb mice. Total RNA was isolated from flushed long bones. (g–i) Real-Time PCR analysis of (g) glycophorin A (h) CD45 (i) CD19 in the flushed long bones and bone marrow. n=4. ^*p < 0.05 versus WT. (j) Immunoblot analysis showing FoxO1 protein levels in bones lysates of wild type and Ctnnb1^CAosb mice. Blot is representative of n=3. Results are mean ± SD. All mice were 1 month of age.