Figure 2

Expression profiles of FGF receptors (FGFRs) in ESFT cell lines and clinical ESFT samples and essential role of FGFR1 in bFGF-induced motility of ESFT cells. (A, left panel) Transcript levels of FGFRs, relative to GAPDH, determined by quantitative real-time PCR in ESFT cell lines. Control of FGFR1, 2 and 3; U20S, FGFR4; MG63. (A, right panel) Representation of protein status of the FGFRs by western blot in various Ewing's sarcoma cell lines. Actin was used as a loading control. (B)The expression profile of ESFT biopsy samples was detected by RT–PCR. GAPDH was used as an internal control. P: positive control. (C) RD-ES cells were incubated with bFGF (20 ng ml⁻¹) for 20 min, and the total cell lysates were subjected to western blot analysis with anti-FGFR1 and anti-tyrosine-phosphorylated FGFR1 antibodies. In RD-ES cells, bFGF induced the tyrosine phosphorylation of FGFR1, and pre-treating RD-ES cells with a specific inhibitor of FGFR1, SU5402 (20 μ M), for 2 h inhibited this phosphorylation. Actin is shown as a loading control. Data are representative of at least three independent trials. (D and E) The effects of SU5402 on growth factor-induced motility of RD-ES cells were assessed by a wound-healing assay (panel D) and a chemotaxis assay (panel E). The concentrations of the respective growth factors in the assays were as follows: bFGF (10 ng ml⁻¹), IGF-1 (20 ng ml⁻¹), and PDGF-BB (20 ng ml⁻¹). The growth factors and SU5402 were added into the lower chamber at the same time. In chemotaxis assay, 2 × 10⁵ cells were plated onto the upper chamber. Experiments were performed in triplicate and repeated at least three times. Data are shown as mean±s.d. ^***P<0.001, ^*P<0.05 vs indicated growth factor with DMSO. #, not significant.