Fig 1.
FGF1 amplifies TGFβ-1-induced EMT in mammary epithelial cells.
Starved MCF10A cells were treated with or without 5 ng/ml TGF-β1 in the absence or presence of 50 ng/ml FGF1 for 48 h. A, Cell lysates were applied for Western blotting with the indicated antibodies. GAPDH and total ERK1/2 were used as a loading control. B, Total RNA was extracted and revers transcribed to cDNA. N-cadherin and vimentin, Snail1 and Snail2 expressions were assessed by real-time RT-PCR. C, Cell lysates were applied for Western blotting with the indicated antibodies. D, Matrigel invasion assay was performed on MCF10A cells following treatment with 5 ng/ml TGF-β1 in the presence or absence of FGF1. E, Conditioned media were applied for gelatin zymography and MMP2 activity were assessed by gelatin digestion in the gel. F, MCF12A cells were starved and treated with or without 5 ng/ml TGF-β1 in the absence or presence of 50 ng/ml FGF1 for 48 h. Western blotting was performed with the indicated antibodies. Data represent the mean ± S.E. (n = 3; *, p < 0.05). Bands intensity was measured by densitometry.
Fig 2.
Integrin αvβ3 and FGFR1 are induced by TGF-β1 in MCF10A cells.
A, Starved MCF10A cells were stimulated with or without 5 ng/ml TGF-β1 for 48 h. Cell surface expression of integrins were determined by FACS analysis using indicated antibodies. B, We performed time-course analysis of FGFR1, FGFR2, and integrin αv and β3 expression by Western blotting. Starved MCF10A cells were stimulated with DMSO or TGF-β1 (5 ng/ml) for the indicated time periods. Cell lysates were applied for Western blotting and proved with antibodies shown in figure. GAPDH was used as loading control. C, Dose-dependent effect of TGF-β1 on integrin αvβ3 was assessed by FACS flow cytometry. MCF10A cells were treated with the indicated concentration of TGF-β1. D, Induction of integrin αvβ3 was determined by immunofluorescence microscopy. Cells were stimulated with TGF-β1 (0.5 or 5 ng/ml) for 48 h, and stained with anti-integrin αvβ3 antibody (LM609) with FITC conjugated secondary antibody to visualize (upper). Lower panels indicate representative phase contrast images of TGF-β1 stimulated cells. Scale bar = 20 μm.
Fig 3.
Integrin αvβ3 is required for FGF1 to amplify TGF-β1-induced EMT.
A, 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. B, 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. C, 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. D, 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. E, 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 C. Protein levels were determined by Western blotting with the indicated antibodies. Data represent the mean ± S.E. (n = 3; *, p < 0.05). Bands intensity was measured by densitometry.
Fig 4.
Integrin binding defective FGF1 mutant R50E is not able to enhance EMT.
A, 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. B, 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; *, p < 0.05, **, p < 0.01). Bands intensity was measured by densitometry.
Fig 5.
FGF1 augments TGF-β1-induced loss of polarity depends on the interaction between FGF1 and integrin αvβ3.
A and B, MCF10A cells were seeded on glass cover slip coated with growth factor-reduced Matrigel. After 2 weeks of incubation, acinar structures were formed on the Matrigel. Assay medium was replaces to DMEM/F12 containing 2% horse serum and 2% growth factor-reduced Matrigel. Then cells were treated for inducting EMT with 5 ng/ml TGF-β1 together with either 50 ng/ml WT-FGF1 (WT) or integrin binding defective FGF1 mutant (R50E) for 4 days. The acini were immunostained with an anti-integrin αvβ3 (A, green) or an anti-integrin α6 (B, green) antibody together with DAPI labels nuclei (blue). Confocal images were acquired at the center plane of the acinar structure. Scale bar = 50 μm. C, MCF10A cells were treated as in A, then acini indicating discontinuous basement membrane was counted from the images.
Fig 6.
FGFR and ERK signaling is required for FGF1 to mediate TGF-β1 induced EMT in MCF10A cells.
A and B, 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.