iluted 1:200 for 20 min and with peroxidase for 10 min. Diaminobenzidine was used as the substrate for color development and visualization under the a Nikon microscope at indicated magnifications. PBS was 
used to replace the primary antibody in negative controls. Bone mineral density measurement Mice were anesthetized with isoflurane and placed in a prone position. The whole body bone mineral density was determined by dual-energy X-ray absorptiometry using software specifically designed for BMD measurement of small animal measurement in vivo. The BMD values were expressed as grams per square centimeter. Bone protein preparation Femoral bone was collected and prepared for protein extraction. In general, the femora of mice were dissected free of all connective tissues, and bone marrow were flushed away then crushed using a mortar and pestle in liquid nitrogen. The lysates were collected in 500 ml RIPA buffer for total protein extraction in presence of Protease Inhibitor Cocktail and PhoSTOP Phosphatase Inhibitor Cocktail. The homogenates were centrifuged at 12,000 g at 4uC for 20 min. Resultant supernatant was quantified by a BCA assay and subjected PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19630074 to Western Blot analysis. Statistical analysis Data are shown as mean 6 SEM of data from at least three separate experiments, each performed with triplicate samples. Cyclin G2 Inhibits Estrogen-Mediated Osteogenesis Statistically analyses were conducted using Student’s t-test, one- or two-way ANOVA followed by appropriate pairwise post hoc tests with correction for multiple comparisons. Differences were considered statistically significant if P,0.05. Results Cyclin G2 is involved in estrogen-regulated osteogenesis in vivo and in vitro In previous studies, cyclin G2 was reported to be downregulated by estrogen, which deficiency is known to induce bone loss and leads to PMOP. We first determined whether cyclin G2 is involved in this process. Ovx mice and Sham mice were generated and femoral bones were isolated for protein extraction and inmmunohistochemical staining. Expression of cyclin G2 proteins in bone tissues of Ovx mice vs. Sham mice were analyzed by Western blot and inmmunohistochemical staining. It was found that expression PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19632393 of cyclin G2 was up-regulated in the bone of Ovx mice, which is known to be Cetilistat site estrogen deficient, accompanied with decreased bone density. This finding suggests a role of cyclin G2 in estrogen-regulated osteogenesis in vivo. We further assessed the expression of cyclin G2 in OS-medium treated C2C12 cells with or without E2. Both mRNA and protein expression of cyclin G2 was reduced by OS-medium treatment, while the inhibition expression stimulated by E2 exhibited milder bands for cyclin G2. We then confirmed ALP activity and calcium accumulation in the osteogenically differentiated C2C12 cells and observed that OS-medium with or without E2 induced C2C12 cells to osteogenesis. These manners were inversely correlated with expression of cyclin G2 and suggest that cyclin G2 has a potential role in the regulation of osteogenesis in vitro, especially when regulated by estrogen. Cyclin G2 suppresses osteogenic differentiation in pluripotent mesenchymal precursor cell line C2C12 To directly address the effect of cyclin G2 in osteogenic differentiation, C2C12 cells was infected with a recombinant retrovirus carrying cyclin G2 for higher and longer expression of ectopic cyclin G2 and induced to osteogenesis. The ectopic expression of cyclin G2 was confirmed by semi-quantitative R
