Figure 4

GDF15 promoted the growth of the ESCC cell lines by activating Akt and Erk1/2 phosphorylation. (a) Effect of GDF15 on proliferation in ESCC cell lines. Cells were treated for 24 h with rhGDF15 (10, 50, and 100 ng/ml) under serum-free conditions. The percentage of cell proliferation in controls versus GDF15-treated cells was measured by Trypan blue dye exclusion assay. The growth of ESCC cells was promoted by rhGDF15 dose-dependently. Data are mean±s.e.m. from five wells (**P<0.01, ***P<0.001). NT, no treatment. (b) Phosphorylation of Akt and Erk1/2 in ESCC cell lines by GDF15. TE-8, TE-9, and TE-15 cells were treated with 100 ng/ml rhGDF15 for 0, 10, 30, and 60 min under serum-free conditions. The total proteins were harvested for western blot analysis using specific antibodies against Akt, p-Akt (Ser473), p-Akt (Thr308), Erk1/2, p-Erk1/2 (Thr202/Tyr204), and β-actin. Not only Erk1/2 but also Akt was activated after 10 min of rhGDF15 stimulation in ESCC cell lines. (c) The growth-promoting effect of GDF15 on ESCC cell lines was inhibited by LY294002 and PD98059. TE-8, TE-9, and TE-15 were pretreated with a PI3K inhibitor (LY294002), a MEK1/2 inhibitor (PD98059), or none for 30 min followed by stimulation with 100 ng/ml rhGDF15 for 24 h under serum-free conditions. The growth activity of the cells was assessed by the MTS assay. GDF15-induced proliferation was inhibited by both LY294002 and PD98059. Data are mean±s.e.m. in triplicate (**P<0.01).