Inhibition of Akt activation in osteoblasts promotes mineralized-matrix production. (A) To determine the concentration range at which AG-1295 and LY294002 were inhibitory to PDGF-induced Akt phosphorylation, serum-deprived cells were treated with a range of drug concentrations and stimulated with PDGF (10 ng/mL) for 10 minutes, after which cell lysates harvested. Equivalent levels of cellular proteins were transferred to PVDF membranes and probed with phosphorylation-specific antibodies as indicated. (B) To determine the effect of the drugs on mineral formation, iliac crest bone trephine explant cultures were grown under osteoinductive conditions in the presence of an inhibitory concentration of the indicated drug for 4 weeks and the concentration of calcium per 105 cells was then quantified. IM represents imatinib; AG, AG-1295; LY, LY294002; and UT, untreated. Mean values are shown; error bars represent the standard error of triplicate measurements.
