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Fig. 1. Localized mechanical stimuli were applied to HUVECs by displacing an FN bead. (A) Schematic drawing of the experimental set-up. A HUVEC was plated on a fibronectin-conjugated elastic polyacrylamide gel substrate (blue). (B) HUVECs were stained with anti-paxillin antibodies (upper panel) and Rhodamine-phalloidin (middle panel), and sectioned in the x-z plane. These images are overlaid in the panel below. The yellow semi-circle shows the position of the FN bead. Stress fibers (yellow arrows) connect the apical focal adhesions (FAs) to the basal FAs. The bottom panel shows the x-y confocal image of the cell. A 10 µm bead promoted formation of focal adhesions on the apical surface (shown by white arrows). The pattern of FAs underneath the FN bead was scattered and irregular (arrowheads). FAs on the cell base were also imaged. Twenty confocal images (5 µm thickness) were projected to the x-y plane. (C) An example of gel deformation caused by a mechanical stimulation. An FN bead (shown by green dotted circle) on a HUVEC (white broken line denotes the cell perimeter) was displaced by a piezoelectric-driven glass pipette (shadows in the top and bottom panels). Small orange circles indicate the position of 50 nm fluorescent beads (middle panel). Insets show fluorescent beads at higher magnification (a fluorescent spot at the centre of the circle; scale bar, 5 µm). Each red arrow in the bottom panel indicates the direction and relative amplitude of fluorescent particle displacement (longest arrow corresponds to 0.76 µm displacement) when the FN bead was moved 4 µm in the direction shown in the top panel by an arrow. (D) Very small substratum deformation underneath the bead was observed in cytochalasin-D-treated cells (note red arrow in the lower right marginal region of the cell). (E) Time course of the displacement of a 50 nm bead exposed to the mechanical stimulus along with the time course of another bead that did not move because it is located far from the FN bead (>40 µm). The interval between frames was 1 second.
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