Fig. 5: CUEDC2 inhibits STAT3-mediated osteogenesis by regulating SOCS3 protein stability.

a–c The level of SOCS1, SOCS3, and phosphorylated STAT3 proteins was evaluated by western blotting. The relative protein levels of the indicated proteins were calculated after normalization to β-ACTIN. *P < 0.05, **P < 0.01 versus control. a MC3T3-E1 cells were cultured for 6 days under the conditions shown in the Fig. 2b legend. b, c MC3T3-E1 cells transfected under the same conditions as the knockdown (Fig. 4a) of CUEDC2. After 24 h, cells were cultured with GM for 1 day. b The cytosolic and nuclear proteins were extracted from the cells for western blotting. MEK2 and LAMIN B were used as a loading control. The interaction between CUEDC2 and SOCS3 proteins (d) and the level of ubiquitin-conjugated SOCS3 proteins (e) were evaluated. HEK293T cells were co-transfected with indicated plasmids, as shown in d, e. Cell lysates were immunoprecipitated with anti-HA (d) or anti-SOCS3 or anti-UB (e) followed by western blotting with the indicated antibodies. As an input, 1% cell lysates were loaded. f, g Infection with Ad-shCUEDC2 or Ad-GFP was performed under the same conditions as described in the legend of Fig. 3b. After infection, the cells were cultured with Stattic (+, 2.5 µM; ++, 5 µM) in the presence of OM to investigate osteoblast marker gene expression (2 days), ALP activity (5 days), and mineralized nodule formation (12 days). **P < 0.01, ***P < 0.001 versus OM. ^##P < 0.01, ^###P < 0.001 versus Ad-shCUEDC2 with OM. f Real-time PCR analysis. g ALP and AR staining.