(A) D0.11.10 cells (3 × 103) were pretreated with wortmannin (0.3 μM), PD98059 (10 μM), SB203580 (10 μM) or SP600125 (10 μM) or 0.1% DMSO vehicle control (CTR) for 30 min prior to treatment with 10 nM ghrelin. After incubation for 24 or 48 hours, the plates were examined for cell proliferation using MTS assay. The data are expressed as the percentage of cell number compared to the CTR group at time 0. Values represent the mean and SEM of determinations made in three independent experiments. *P<0.05, **P < 0.01 compared to 0 h (control); #P< 0.05, ##P < 0.01 compared to ghrelin treatment. (B) D0.11.10 cells (2 × 106) were cultured with 10 nM ghrelin in the presence or absence of various concentrations of wortmannin or PD98059 for 30 min after which the cells were lysed and examined for Akt and ERK1/2 phosphorylation via immunoblot analysis. These blots are representative of results obtained in 5 separate experiments.