BMP2 and GSK126 synergize to induce expression of osteogenic genes

BMP2 and GSK126 synergize to induce expression of osteogenic genes Overall design: 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 a-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 ß-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).