JCS022053 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Effects of cytochalasin D and colcemid on cytoskeletal architecture in cultured HUVECs. After a 30 minute incubation in normal growth medium (a,b), plus 100 nM cytochalasin D (c,d) or plus 1µM colcemid (e,f), cells were fixed with 4% paraformaldehyde, 137 mM NaCl, 8 mM Na₂HPO₄, 1.5mM KH₂PO₄, 5 mM MgCl₂ and 2 mM EGTA for 15 minutes, and permeabilized in acetone at -20°C for 5 minutes. Actin filaments were stained with Rhodamine-phalloidin (a,c,e), and microtubules with the anti-β-tubulin monoclonal antibody Tub 2.1 (ICN Biomedicals) (b,d,f). Cytochalasin D nearly perfectly disassembled actin stress fibers (c), but did not affect microtubules (d). By contrast, colcemid largely disassembled microtubules (f), but not actin stress fibers (e).

• Supplemental Figure S2 -

  Fig. S2. Ca²⁺ transients induced by bead displacement under different conditions. FN bead displacement (1 µm for 100 milliseconds) did not induce Ca²⁺_i increase in nominally Ca²⁺-free solution (asterisks), and in solution contained 20 µM Gd³⁺ (open squares) or 100 nM cytochalasin D (closed squares). No Ca²⁺_i increase was also observed by the displacement of a bead conjugated with anti-LDL antibody (open circles) or with nonactivating anti-integrin antibody (K-20) (closed circles) in normal solution. Ca²⁺_i increases were induced only when the FN bead was displaced in normal solution (closed triangles).

• Supplemental Figure S3 -

  Fig. S3. Ca²⁺ transient from top and bottom membrane of HUVECs by the mechanical stimulation. (A) A schematic presentation of the experimental setup. HUVECs were loaded with Fluo3-AM and then line-scan images of Ca²⁺ increase were obtained at the top, middle or bottom of the cell. Fluorescence image of Fluo-3 and displacement of bead were imaged simultaneously to examine the transient Ca²⁺ increase just after the mechanical stimulation; intensity of red color denotes the Fluo-3 fluorescence signal and green color denotes the position of the FN bead. Time-lapse line scans (4.3 milliseconds/line) were made from the top (B), middle (C) and bottom (D) of the cell; the horizontal axis denotes the line scan and the vertical axis denotes time from top to bottom. Yellow triangles indicate the time of stimulation. Averaged fluorescence intensity of Fluo-3 inside the yellow rectangle was analyzed. Graph E shows relative fluorescence intensity changes (mean ± s.e.m., n=3-6) of fluo-3 at the top (green line), middle (blue line) and bottom (red line) of the cell. Ca²⁺ transient starts at top and bottom of the cells within 4.3 milliseconds of the stimulation, followed by the Ca²⁺ transient in the middle of the cells.

• Supplemental Figure S4 -

  Fig. S4. HUVECs were labeled with Alexa Fluor 546-conjugated anti-β1-integrin antibody (Kawakami et al., 2001) and stained anti-paxillin antibodies after fixation. Left panel is a DIC image of a cell with a 10 µm FN bead. Center and right panels are fluorescence images of anti-β1-integrin and anti-paxillin staining.