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Fig. 12. Re-expression of caveolin-1 rescues endocytosis of β1 integrin in caveolin-1 siRNA cells. FN-null myofibroblasts expressing caveolin-1 siRNA (shcav) were transduced with Ad-cav or Ad-tet adenoviruses. (A-I) Cells were incubated with 10 µg/ml TR-fibronectin overnight. Cells were washed, and then incubated for 8 hours in cell culture medium lacking fibronectin, but containing 50 µM chloroquine. Cells were stained with anti-β1 integrin antibody (FITC). Upper panels, shcav without virus transduction; middle panels, Ad-cav; lower panels, Ad-tet. A,D,G: TR-fibronectin; B,E,H: β1 integrin; C,F,I, overlay images. Arrows in D show the internalized fibronectin; arrows in E show intracellular β1 integrins; arrows in F show colocalized fibronectin and β1 integrins. D`, E` and F` are enlarged images of area shown by the rectangle in F. (J-M) In the absence of fibronectin, cells were incubated with 50 µg/ml antibodies to 5 integrin (J) or β1 integrin (K-M) at 4°C for 45 minutes. Cells were then processed for integrin endocytosis assay. (J) The fluorescence intensity of endocytosed 5 integrin was quantified using a MATLab-based program. Graph shows the relative fluorescence intensity of endocytosed 5 integrin in shcav cells with or without virus transduction. Intracellular fluorescence intensity of shcav cells without virus transduction was set equal to 1 (mean ± range from two independent experiments). All images are optical sections collected by confocal microscopy. Scale bars: 20 µm.

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