Figure 1
Inhibitory effect of ZSTK474 on OC formation. Mouse bone marrow cells containing OC precursors were cultured with osteoblasts in the presence of 1α,25-(OH)2D3 for five days. Indicated concentrations of ZSTK474, LY294002, AS605240 (a PI3-Kγ-selective inhibitor), or IC87114 (a PI3-Kδ-selective inhibitor) were added at the initiation of cultures. Mouse bone marrow cells were also cultured with M-CSF (10 ng/ml) for two days and then with M-CSF and sRANKL (100 ng/ml) for another three days. The inhibitors were added simultaneously with sRANKL. a) and c) TRAP-positive multinucleated cells were counted as OC. The columns and bars indicate the mean and standard deviation (S.D.) from duplicated wells. b) OC formation in co-culture with osteoblast (upper) and culture with M-CSF and sRANKL (lower). Representative results were shown in a, b, and c. d) RANKL mRNA was measured using real-time PCR, with results normalized the value of β-actin. The columns and bars indicate the mean and S.D. of three independent experiments. * = P > 0.05, ** = P < 0.01, *** = P < 0.001.