Supplemental Figure S1
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Fig. S1: Quantification of adhesion complexes on FN-coated surfaces and fibrillar FN matrices. HT1080 cells were plated on FN-coated coverslips (left column), uncrosslinked FN matrix (middle column) or 4.5 kPa FN matrix crosslinked with 10% formaldehyde (right column) for 3 hours. Unspun samples (top row) or samples after spinning (Second and third rows) were stained using an anti-vinculin monoclonal antibody (Sigma) followed by fluorescein-conjugated goat anti-mouse antibodies (Molecular Probes). Images were captured for unspun, minimal shear (∼0 dynes/cm2), and maximal shear (250 dynes/cm2) samples on all three substrates. Boxed areas are magnified in the insets and different sized adhesions are labeled as indicated in the graph (1, 2, 3; bottom left). Sizes of vinculin-positive adhesions were quantified by thresholding images to a pixel intensity of 50 a.u. which excluded peri-nuclear staining but included adhesions in the thresholded images. Image features of 0.15 µm2 (∼20 pixels) and larger were measured and counted for each cell and displayed as a histogram. Values are average ± standard deviation determined from more than 10 cells/condition. Scale bars: 20 µm.
