|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 9. (A-F) Caveolin-1 regulates short-term fibronectin endocytosis. Cells expressing caveolin-1 siRNA (shcav, A-C) or control siRNA (shluc, D-F) were incubated with 10 µg/ml AF488-fibronectin and 50 µg/ml TR-conjugated 9EG7 at 4°C for 1 hour during the pulse. After 30 minutes of chase at 37°C, cells were incubated with 0.2% Trypan Blue for 3 minutes to quench extracellular fluorescence. AF488-fibronectin, A,D; TR-9EG7, B,E; overlay images, C,F. Scale bar: 10 µm. (G) Flow cytometric analysis of endocytosed fibronectin. Cells expressing caveolin-1 siRNA (shcav) or control siRNA (shluc) were incubated with 10 µg/ml AF488-fibronectin at 4°C for 1 hour in the presence or absence of 1 mg/ml unlabeled fibronectin. After 30 minutes of chase, cells were processed for flow cytometry. The specific MFI was determined by subtracting the total MFI (shcav, 315; shluc, 489) from the MFI of cells incubated with unlabeled fibronectin (shcav, 147; shluc, 177). Data show the fold change relative to the MFI of endocytosed AF488-fibronectin in control cells, which was set equal to 1 (mean ± range). (H-J) Colocalization of internalized fibronectin with lipid raft marker. FN-null myofibroblasts were incubated with 10 µg/ml TR-fibronectin and 2 µg/ml AF488-CTxB at 4°C for 1 hour. After 2 hours of chase, cells were incubated with 0.2% Trypan Blue for 3 minutes and then processed for imaging assay (H, TR-fibronectin; I, AF488-CTxB; J, overlay image, arrowheads point to colocalized TR-fibronectin and AF488-CTxB). (K-O) Colocalization of internalized fibronectin, β1 integrins and caveolin-1. FN-null myofibroblasts were incubated with 10 µg/ml TR-fibronectin overnight. Cells were washed and then incubated for 12 hours in cell culture media lacking fibronectin, but containing 50 µM chloroquine. Cells were stained with anti-β1-integrin and anti-caveolin-1 antibodies, followed by FITC-conjugated anti-hamster and Alexa Fluor 660-conjugated anti-rabbit secondary antibodies. (K, TR-fibronectin; L, β1 integrin; M, caveolin-1; N, overlay image, arrowheads show the colocalization of triple colors). Fluorescence intensity profiles (O) were generated using ImageJ software (National Institutes of Health, Bethesda, ML). A line was manually drawn to cross several TR-fibronectin containing vesicles (as shown in K) and the fluorescence intensity profiles were obtained from each individual color channel. The plot in O was generated in Excel (red, TR-Fibronectin; green, β1 integrin; blue, caveolin-1). All images are optical sections collected from a confocal microscope. Scale bar: 10 µm.
|
