Fig. 2.

FGF signaling and response to FGF2 in chordoma cells. (A) JHC7, UCH1 and UCH2 cells were seeded in six-well plates for 24h, then were starved overnight and treated with FGF2 (100ng/ml) for 5, 10 and 20min. Cell protein extracts were used for western blot analysis of FGFR2 and its phosphorylation (p-FRS2-α). Representative bands from three independent experiments are shown. (B) JHC7, UCH1 and UCH2 cells were seeded in six-well plates for 24h, then were starved overnight and treated with FGF2 (10ng/ml) for 24h. Cell protein extracts were used for western blot analysis of FGFR2, p-FRS2-α, MEK 1/2, ERK 1/2, p-MEK 1/2 and p-ERK 1/2. Representative bands from three independent experiments are shown. (C) JHC7, UCH1 and UCH2 cells were starved overnight and treated with FGF2 (10ng/ml) for 24h. Cells were fixed and double immunofluorescence stained for p-ERK 1/2 with anti-p-ERK 1/2 (green) and for brachyury with anti-brachyury (red). Meanwhile, a negative control from JHC7 cells incubated second antibodies alone was included. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). Yellow color indicates the co-localization of p-ERK 1/2 and brachyury. Scale bars = 50 µm. Representative images from three independent experiments are shown.