Cell signaling pathways activated upon FGF-10 stimulation and their impact on cell behavior. (A) Serum-starved MCF-7 cells were stimulated with 100 ng/ml FGF-10 in the presence of 7.5 µg/ml heparin in serum-free media for 15, 30, and 60 min. Where indicated, cells were pretreated with 2 µM PD173074 (1 h). Cell lysates were prepared, and Western blotting was performed using different primary antibodies. Stimulation with FGF-10 activated the FRS2–ERK pathway as well as the AKT pathway. FGF-10 treatment triggered rapid ERK phosphorylation, which was blocked by the FGFR inhibitor PD173074. Other pathways (PLC-γ and PI3K) were also investigated, but although FGF-10 induced AKT phosphorylation, this was not abrogated by treatment with PD173074. PLC-γ was not activated by FGF-10 treatment. (B) Migration of MCF-7 and MDA-MB-231 cells was monitored using 8-µm-pore Transwell migration filters in serum-free medium in the presence of FGF-10/heparin (100 ng/ml and 7.5 µg/ml, respectively) and/or 2 µM PD173074. Cells were allowed to migrate overnight. FGF-10 treatment increased Transwell migration significantly, and this effect was blocked in the presence of the PD173074. (C) Immunostaining with an anti-Ki67 antibody to quantify cell proliferation after 48 h revealed that treatment with FGF-10 resulted in a significant increase in the percentage of positive cell staining. (D) FGF-10 treatment reduced apoptosis in MCF-7 cells after 48-h treatment with FGF-10, as determined by TUNEL assay (PI, propidium iodide). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (ANOVA for B; Student’s t test for C and D). Bars, 25 µm. Error bars show means ± SEM.