JCS014977 Supplementary Material

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• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. Soluble fibronectin endocytosis in β1 null cells. (A) GD25 (β1 null) and GD25β1 (β1 re-expressed) cells were incubated with 10 µg/ml AF488-fibronectin at 4°C for 1 hour. Cells were then processed for fibronectin endocytosis pulse-chase assay. After 1 hour of chase at 37°C, cells were processed for flow cytometry to quantify internalized fibronectin as described in the Materials and Methods. The numbers next to the peak are the MFI of internalized AF488-fibronectin. The levels of internalized AF488-fibronectin were indistinguishable between GD25 and GD25β1 cells, and thus, only one peak is seen in the overlay. (B-C) RGD peptides block soluble fibronectin endocytosis in GD25 cells. GD25 cells were seeded in serum-free medium with 25 µg/ml cycloheximide on vitronectin-coated dishes. Cells were allowed to adhere for 8 hours and were then incubated with 0.2 mg/ml RGD (B) or RGE peptides (C) at 4°C for 30 minutes. 10 µg/ml AF488-fibronectin was added to the medium, and cells were processed for fibronectin endocytosis pulse-chase assay. After 2 hours of chase, cells were incubated with 0.2% Trypan Blue for 3 minutes, and then fixed for imaging assay. Blue, DAPI. Scale bar: 10 µm.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Integrin endocytosis in caveolin-1-knockdown cells. Cells expressing caveolin-1 siRNA (shcav) or control cells (shluc) were incubated with 10 µg/ml TR-fibronectin overnight. Cells were washed and then incubated with 9EG7 (25 µg/ml) at 4°C for 45 minutes. Following washing, cells were incubated for 3 hours with cell culture medium lacking fibronectin, but containing 125 µM chloroquine. Cells were stained with a FITC-conjugated goat anti-rat secondary antibody. Upper panels, shcav; lower panels, shluc. (A,D) 9EG7; (B,E) TR-fibronectin; (C,F) overlay images. Scale bar: 10 µm.

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. Fibronectin matrix after detergent extraction of fibronectin-null myofibroblasts. Pre-assembled fibronectin matrix was prepared as described in the Materials and Methods. (A) TR-fibronectin matrix before detergent extraction; (B) Fibronectin matrix following detergent extraction of cells. Representative images are shown. Scale bar: 40 µm.