Figure 4.

Activation of PI3K/AKT is required for BMP2-induced osteogenic differentiation. (A) C3H10T1/2 cells were cultured in osteogenic medium containing BMP2 at various doses (0, 50, 100 or 200 ng/ml) for 48 h. The expression of pAKT and total AKT was examined using western blotting. Densitometry for the ratio of pAKT/AKT protein bands was analyzed using ImageJ. Data are representative of 3 independent experiments. (B) C3H10T1/2 cells were cultured in osteogenic medium containing BMP2 (0 and 200 ng/ml) with/without LY294002 (10 µM) for 48 h. The expression of pAKT and AKT was examined using western blotting. Densitometry for the ratio of pAKT/AKT protein bands was analyzed using ImageJ. Data are representative of 3 independent experiments. (C) ALP activity assay in C3H10T1/2 cells cultured in BMP2 (200 ng/ml)-containing osteogenic medium, treated with bpV(pic) (10 µM) and/or LY294002 (10 µM) for 3 days (n=3). (D) Enzyme-linked immunosorbent assay for osteocalcin secretion in supernatant harvested from C3H10T1/2 cells cultured in osteogenic medium for 7 days (n=3). *P<0.05; **P<0.01. BMP, bone morphogenetic protein; pAKT, phosphorylated AKT; ALP, alkaline phosphatase.