FGFR and ERK signaling is required for FGF1 to mediate TGF-β1 induced EMT in MCF10A cells.

<p><i>A and B</i>, Starved MCF10A cells were stimulated with 5 ng/ml TGF-β1 and 50 ng/ml FGF1 in the presence of 1 μM PD173074 specific inhibitor of FGFR1 (A) or 10 μM U0126 specific inhibitor of MEK1 (B) for 48 h. DMSO was used as a solvent control for the chemicals. Cells were then lysed, and protein levels were analyzed by Western blotting with the indicated antibodies. Bands intensity was measured by densitometry.</p

Integrin binding defective FGF1 mutant R50E is not able to enhance EMT.

<p><i>A</i>, Starved MCF10A cells were treated with 5 ng/ml TGF-β1 in the presence of 50 ng/ml WT-FGF1 (WT) or integrin binding defective FGF1 mutant (R50E) for 48 h. Cell lysates were processed for Western blotting with the indicated antibodies. ERK1/2 and GAPDH served as a loading control. <i>B</i>, Starved MCF10A cells were treated with 5 ng/ml TGF-β1 in the presence of 50 ng/ml WT-FGF1 (WT) or integrin binding defective FGF1 mutant (R50E) for 24 h. Confluent cell monolayer was scratched by pipet tip and cultured additional 24 h in same conditions. Images were taken by phase-contrast microscopy and measured area of wound closure. Data represent the mean ± S.E. (n = 3; *, <i>p</i> < 0.05, **, <i>p</i> < 0.01). Bands intensity was measured by densitometry.</p

Integrin αvβ3 is required for FGF1 to amplify TGF-β1-induced EMT.

<p><i>A</i>, MCF10A cells were transfected with siRNA designed to target integrin β3 (si β3#1 and si β3#2). Non-targeting scramble siRNA (scr) is also transfected as a control. 24h post-transfection, cells were treated with 5 ng/ml TGF-β1 in the absence or presence of FGF1 (50 ng/ml) for 48h. Cells were then lysed, and protein levels were analyzed by Western blotting and probed with the indicated antibodies. <i>B</i>, MCF10A cells were transfected and treated same as above, confluent cells were then scratched by pipet tip, which allowed cells to migrate to the open space and cultured for additional 24h. Graph shows the fold change of closed wound area after 24 h. <i>C</i>, SK-BR-3 breast cancer cells stably transfected with shRNA for either integrin β3 (sh β3#5 and sh β3#12) or non-targeting scramble shRNA (scr), then cells were stimulated with 50 ng/ml FGF1 for 24h. Protein levels were determined by Western blotting with the indicated antibodies. <i>D</i>, SK-BR-3 cells were transfected and treated same as above, confluent cells were scratched and cultured for additional 24h. Graph shows the fold change of closed wound area after 24 h. <i>E</i>, ZR-75-30 breast cancer cells were stably transfected with integrin β3 expression vector or empty vector as a control, then cells were stimulated same as <i>C</i>. Protein levels were determined by Western blotting with the indicated antibodies. Data represent the mean ± S.E. (n = 3; *, <i>p</i> < 0.05). Bands intensity was measured by densitometry.</p

R50E suppresses WT FGF1- induced tube formation of endothelial cells in vitro.

<p>Serum starved HUVECs were plated on Matrigel-coated plates, and incubated in WT FGF1 (5 ng/ml) or the mixture of WT FGF1 (5 ng/ml) and R50E (250 ng/ml) for 8 h. a. Representative tube formation images are shown. Scale bar = 200 µm. b. The number of branch points was counted per field from the digital images. Data is shown as means +/− SE. Statistical analysis was done by one-way ANOVA plus Tukey analysis.</p

R50E suppresses WT FGF1-induced angiogenesis in rat aortic ring.

<p>Isolated rat aortic ring was embedded in collagen gels in DMEM containing WT FGF1 (50 ng/ml), R50E (50 ng/ml) or the mixture of WT FGF1 (50 ng/ml) and R50E (2500 ng/ml) and cultured for 10 days. Representative phase contrast images of 3 independent experiments are shown. Scale bars, 100 µm.</p

R50E suppresses FGF1- and FGF2-induced angiogenesis (branching formation) in CAM models.

<p>Saline- or FGF- impregnated filter disks are placed on blood vessels in otherwise avascular sections of CAM (day 11) to induce angiogenesis. The disks and underlying CAM tissue (day 13) are then harvested. Neovascularization was then scored by counting vessel branches present in the CAM tissue below the filter from digital images. a) and b) Quantification of dose response. Five ng/ml is optimum, c) Suppression of FGF1-induced angiogenesis by excess R50E. d) Suppression of FGF2-induced angiogenesis by excess R50E. The data suggest that R50E suppresses FGF1- and FGF2-induced angiogenesis in the CAM model. Statistical analysis was done by one-way ANOVA plus Tukey analysis.</p

R50E suppresses WT FGF1-induced endothelial cell migration.

<p>Lower side of the filter in the modified Boyden chamber was coated with fibronectin (10 µg/ml). The lower chamber was filled with serum-free medium with WT FGF1 (5 ng/ml) or the mixture of WT FGF1 and excess R50E (5 and 250 ng/ml, respectively). HUVECs were plated on the filter and incubated for 6 h. Chemotaxed cells were counted from the digital images of the stained cells. Data is shown as means +/− SE per field. Statistical analysis was done by one-way ANOVA plus Tukey analysis.</p

R50E suppresses angiogenesis in Matrigel plug assays in rat.

<p>Matrigel plug containing WT FGF1 (1 µg/ml), R50E (1 µg/ml) or the mixture of WT FGF1 (1 µg/ml) and excess R50E (50 µg/ml) were injected subcutaneously into the back of rat, respectively. The plugs (n = 4−5) were removed 10 days after injection and tissue sections were stained for von Willebrand factor, a blood vessel marker. a. Representative images are shown. Scale bar = 50 µm. b. The number of extended blood vessels were counted under a light microscope. Data is shown as means +/− SE. Statistical analysis was done by one-way ANOVA plus Tukey analysis.</p