BMP2 and GSK126 synergize to induce expression of osteogenic genes

BMP2 and GSK126 synergize to induce expression of osteogenic genes MC3T3 sc4 pre-osteoblasts were treated with BMP2 and GSK126 (Ezh2 inhibitor). Our analysis shows that BMP2 and GSK126 synergistically active expression of osteogenic genes. MC3T3 sc4 cells were maintained in α-minimal essential medium without ascorbic acid (Gibco) containing 10% fetal bovine serum (Atlanta Biologicals), 100 units/ml penicillin and 100 μg/ml streptomycin (Gibco). Cells were seeded in maintenance medium at a density of 10,000 cells/cm2. Next day, cells were treated with vehicle, 5µM GSK126 (Xcess Biociences Inc.), 10 and 50ng/ml BMP2 (R&D Systems), and combination of GSK126 and BMP2 in osteogenic medium (maintenance medium supplementend with 50μg/ml ascorbic acid (Sigma) and 4mM β-glycerol phosphate (Sigma)). Three days later, old osteogenic medium was replaced with a fresh batch of osteogenic medium supplemented with vehicle, GSK126, BMP2, and combination of GSK126/BMP2. Total RNA was isolated on days one and six of osteogenic differentiation using the Direct-zol™ RNA kit (Zymo Research) and TRIzol reagent (Invitrogen).

BMP2与GSK126协同诱导成骨基因表达：将MC3T3 sc4前成骨细胞用BMP2和GSK126（Ezh2抑制剂，Ezh2 inhibitor）处理。本研究分析表明，BMP2与GSK126可协同激活成骨基因的表达。
MC3T3 sc4细胞培养于不含抗坏血酸的α-最低必需培养基（α-minimal essential medium，Gibco）中，该培养基添加10%胎牛血清（Atlanta Biologicals）、100单位/ml青霉素及100μg/ml链霉素（Gibco）。细胞以10,000个/cm²的密度接种于维持培养基中。
次日，将细胞分别用溶剂对照、5µM GSK126（Xcess Biociences Inc.）、10和50ng/ml的BMP2（R&D Systems）以及GSK126与BMP2的联合处理液，在成骨培养基中进行处理；所述成骨培养基为在维持培养基中补充50μg/ml抗坏血酸（Sigma）与4mM β-甘油磷酸（Sigma）制备所得。
处理3天后，更换为新鲜配制的成骨培养基，仍分别添加溶剂对照、GSK126、BMP2及GSK126/BMP2联合处理液。在成骨分化的第1天和第6天，使用Direct-zol™ RNA提取试剂盒（Zymo Research）及TRIzol试剂（Invitrogen）分离总RNA。