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First published online 27 November 2007
doi: 10.1242/jcs.010744

Journal of Cell Science 120, 4388-4394 (2007)
Published by The Company of Biologists 2007
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Neural cell adhesion molecule regulates the cellular response to fibroblast growth factor

Chiara Francavilla1, Sébastien Loeffler1,*, Daniele Piccini1, Angelika Kren2, Gerhard Christofori2 and Ugo Cavallaro1,{ddagger}

1 IFOM-FIRC Institute of Molecular Oncology, I-20139 Milano, Italy
2 Institute of Biochemistry and Genetics, Department of Clinical-Biological Sciences, Center of Biomedicine, University of Basel, Switzerland


Figure 1
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  		Fig. 1. NCAM and FGFR1 colocalize at the cell surface. (A,B) NIH-3T3 cells were co-stained with antibodies against NCAM (red) and FGFR1 (green), followed by fluorochrome-conjugated secondary antibodies. After fixation, cells were subjected to TIRF analysis as described in the Materials and Methods. (B) Magnification of the boxed area in A. Arrows indicate the widespread, tight association of NCAM with FGFR1. Bars, 10 µm (A) and 0.2 µm (B).

 

Figure 2
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  		Fig. 2. NCAM is required for the regulation of the cellular response to FGF. (A) At 48 hours after transfection either with a control siRNA, or with siNCAM1 or siNCAM3, NIH-3T3 cells were left untreated or were stimulated with 5 or 10 ng/ml FGF2 for 10 minutes. After the treatment, cells were lysed, subjected to SDS-PAGE and immunoblotting for phospho-ERK1/2, followed by stripping and immunoblotting for total ERK1/2 and NCAM. After longer exposure, the activation of ERK1/2 with 5 ng/ml FGF2 became detectable also in control cells (not shown). (B) Cells transfected with either a control siRNA or with siNCAM1 were left untreated or were treated with FGF2 for the indicated time periods. Cell proliferation was assessed as described in the Materials and Methods. *P<0.005, relative to untreated control cells.

 

Figure 3
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  		Fig. 3. NCAM is sufficient for the regulation of the cellular response to FGF. (A) L cells stably transfected with empty vector (L-mock), full-length NCAM or {Delta}FN2 were stimulated for 10 minutes with FGF2, followed by cell lysis and immunoblotting for phospho-ERK1/2 and then for total ERK1/2. (B) L cells stably transfected with empty vector, full-length NCAM or {Delta}FN2 were left untreated or were treated with FGF2 for the indicated time periods. Cell proliferation was assessed as described in the Materials and Methods. The growth curves of untreated L-NCAM and L-{Delta}FN2 cells, which were very similar to that of untreated L-mock cells (see also supplementary material Fig. S4A), have been omitted for simplicity. *P<0.005, relative to untreated L-mock cells.

 

Figure 4
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  		Fig. 4. NCAM prevents the binding of FGF to FGFR. (A,B) L cells stably transfected with an empty vector (L-mock) or with NCAM were subjected to the 125I-FGF2 binding assay. Specific binding to low-affinity HSPGs (A) and to high-affinity FGFR (B) was determined as described in the Materials and Methods. (C) Non-transfected L cells were subjected to binding assay with 20 ng/ml 125I-FGF2 in the presence of either FGL or FGLmut at the indicated concentrations. Specific binding to high-affinity FGFR was determined as described in the Materials and Methods, and is represented as the percentage of 125I-FGF2 binding in the absence of peptides. *P<0.005, relative to cells incubated with FGLmut.

 

Figure 5
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  		Fig. 5. The FGL peptide inhibits FGF-induced cell proliferation in a competitive manner. (A) L cells were left untreated or were treated for 0-96 hours with 20 ng/ml FGF2, either without peptides or in the presence of FGL or FGLmut at 50 µg/ml. Cell proliferation was assessed as described in the Materials and Methods. *P<0.005, relative to FGF2-treated cells. (B) L cells were stimulated for 48 hours with FGF2 at 10, 20, 50 or 100 ng/ml in the presence of 10, 20, 50 or 100 µg/ml FGL. For each curve (i.e. for each FGF2 concentration), the data are presented as the percentage of mitogenic activity of FGF2 in the presence of FGL in respect to the mitogenic activity of FGF2 alone. (C) L cells were left untreated or were stimulated for 10 minutes with FGF2, either in the absence or presence of 50 µg/ml FGL, followed by cell lysis and immunoblotting for phospho-ERK1/2 and then for total ERK1/2.

 

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© The Company of Biologists Ltd 2007