Figure 4. Cyclin G2 inhibits C2C12 cells osteogenic differentiation through antagonizing Wnt/β-catenin Signaling.

(A) Western Blot analysis of β-catenin, cyclin D1 and cyclin G2 expression in OS-medium induced C2C12 cells. (B) Western Blot analysis of β-catenin expression in nuclear fraction in C2C12 cells induced by OS-medium. (C) Western Blot analysis of β-catenin, c-Myc and cyclin D1 in cyclin G2-overexpressed C2C12 cells. C2C12 cells were transfected with cyclin G2 expression vector for 48 h, and harvest for total protein extraction followed by Western Blot analysis. (D) Western Blot analysis of β-catenin expression in cyclin G2-overexpressed C2C12 cells that treated with LiCl or NaCl as a negative control. C2C12 cells were transfected with cyclin G2 expression vector, then induced in OS-medium exposed to LiCl (2.5 mM) or NaCl (150 mM). Total protein was harvest after 48 h of induction for Western Blot analysis. (E) Semi-quantitative RT-PCR analysis of osteogenesis differentiation markers (Runx2, Alp, Oc and Opg) in cyclin G2-overexpressed C2C12 cells that treated with LiCl or NaCl as a negative control. Total RNAs were extracted from C2C12 cells after transfected and treated as in D to perform Semi-quantitative RT-PCR analysis. The relative integrated density of each band was digitized by Quantity One. Results are shown as mean ± SEM of data at least three separate experiments, each performed with triplicate samples. *P<0.05, **P<0.01 and ***P<0.001 vs. control groups by two-way ANOVA. (F and G) ALP activity (Results are shown as mean ± SEM of data at least three separate experiments, each performed with triplicate samples. *P<0.05 by one-way ANOVA) and mineralization (ARS staining) of cyclin G2-overexpressed C2C12 cells that treated with LiCl or NaCl. C2C12 cells were infected with recombinant retrovirus carrying cyclin G2 cDNA for 42 h and then treated as in D, followed the measurement of ALP activity on day 7 and calcium deposition on day 14.