Figure 8.
Figure 8. Inhibition of FGFR1 recycling represses NCAM-induced cell migration. (A) HeLa cells cotransfected with HA-FGFR1 (red) and either Rab11-GFP or dn-Rab11–GFP (green) were processed as for Fig. 3 A. Arrowheads indicate the colocalization of Rab11-GFP with HA-FGFR1 (yellow staining), whereas arrows indicate the lack of colocalization of HA-FGFR1 with dn-Rab11–GFP. Asterisk indicates a Rab11-GFP–expressing cell with recycled HA-FGFR1, whereas # shows a dn-Rab11–GFP-expressing cell with no recycling of HA-FGFR1. (B) HeLa cells were transfected with control siRNA (top) or with a mixture of siRNA targeting the Rab11 family (bottom) before transfection with HA-FGFR1 (green) and processing as for Fig. 3 A. Arrows indicate cells transfected with anti-Rab11 siRNA with no recycling of HA-FGFR1 upon FGL stimulation, whereas the asterisk shows a control cell where HA-FGFR1 has recycled to the cell surface. Bars, 10 µm. (C, left) HeLa cells transfected with either Rab11-GFP or dn-Rab11–GFP were stimulated with FGF-2, FGL, or EGF, and the migration of GFP-positive cells in modified Boyden chambers was measured. (right) HeLa cells were transfected with either control or anti-Rab11 siRNA and stimulated and subjected to migration assay as described for the left panel. *, P < 0.005 relative to cells transfected with either Rab11-GFP (left) or control siRNA (right) and stimulated with FGL. (D) HeLa cells transfected with either Rab11-GFP or dn-Rab11–GFP were stimulated with FGL (top) or FGF-2 (bottom) for the indicated time lengths. Lysates from FGL-stimulated cells were immunoblotted for phospho-Src and total Src, whereas lysates from FGF-2–stimulated cells were immunoblotted for phospho-Erk1/2 and total Erk1/2. (E) HeLa cells transfected with either control or anti-Rab11 siRNA were stimulated with FGF-2 or FGL for the indicated time lengths. Knockdown was verified by immunoblotting for Rab11a/Rab11b and for Rab25 using vinculin as a loading control (left). Lysates from stimulated cells were immunoblotted for activated Src or Erk1/2 as described for D. (F) HeLa cells were cultured at 37 or 16°C, stimulated with FGF-2, FGL, or EGF, and subjected to migration assay in modified Boyden chambers. *, P < 0.005 relative to cells grown at 37°C and stimulated with FGL. (G) HeLa cells stimulated with FGF-2, FGL, or EGF in the presence of either DMSO or monensin (10 or 100 µM) were subjected to migration assay in modified Boyden chambers. *, P < 0.005 relative to DMSO-treated cells stimulated with FGL. Data represent the mean ± SEM from three independent experiments.

Inhibition of FGFR1 recycling represses NCAM-induced cell migration. (A) HeLa cells cotransfected with HA-FGFR1 (red) and either Rab11-GFP or dn-Rab11–GFP (green) were processed as for Fig. 3 A. Arrowheads indicate the colocalization of Rab11-GFP with HA-FGFR1 (yellow staining), whereas arrows indicate the lack of colocalization of HA-FGFR1 with dn-Rab11–GFP. Asterisk indicates a Rab11-GFP–expressing cell with recycled HA-FGFR1, whereas # shows a dn-Rab11–GFP-expressing cell with no recycling of HA-FGFR1. (B) HeLa cells were transfected with control siRNA (top) or with a mixture of siRNA targeting the Rab11 family (bottom) before transfection with HA-FGFR1 (green) and processing as for Fig. 3 A. Arrows indicate cells transfected with anti-Rab11 siRNA with no recycling of HA-FGFR1 upon FGL stimulation, whereas the asterisk shows a control cell where HA-FGFR1 has recycled to the cell surface. Bars, 10 µm. (C, left) HeLa cells transfected with either Rab11-GFP or dn-Rab11–GFP were stimulated with FGF-2, FGL, or EGF, and the migration of GFP-positive cells in modified Boyden chambers was measured. (right) HeLa cells were transfected with either control or anti-Rab11 siRNA and stimulated and subjected to migration assay as described for the left panel. *, P < 0.005 relative to cells transfected with either Rab11-GFP (left) or control siRNA (right) and stimulated with FGL. (D) HeLa cells transfected with either Rab11-GFP or dn-Rab11–GFP were stimulated with FGL (top) or FGF-2 (bottom) for the indicated time lengths. Lysates from FGL-stimulated cells were immunoblotted for phospho-Src and total Src, whereas lysates from FGF-2–stimulated cells were immunoblotted for phospho-Erk1/2 and total Erk1/2. (E) HeLa cells transfected with either control or anti-Rab11 siRNA were stimulated with FGF-2 or FGL for the indicated time lengths. Knockdown was verified by immunoblotting for Rab11a/Rab11b and for Rab25 using vinculin as a loading control (left). Lysates from stimulated cells were immunoblotted for activated Src or Erk1/2 as described for D. (F) HeLa cells were cultured at 37 or 16°C, stimulated with FGF-2, FGL, or EGF, and subjected to migration assay in modified Boyden chambers. *, P < 0.005 relative to cells grown at 37°C and stimulated with FGL. (G) HeLa cells stimulated with FGF-2, FGL, or EGF in the presence of either DMSO or monensin (10 or 100 µM) were subjected to migration assay in modified Boyden chambers. *, P < 0.005 relative to DMSO-treated cells stimulated with FGL. Data represent the mean ± SEM from three independent experiments.

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