Migration of A375-SM cells in microchannels. (A–F) A375-SM cells were induced to migrate in microchannels coated with VCAM-1 (A–D) or fibronectin (E–F). (A) Migration velocity of A375-SM cells as a function of VCAM-1–coating concentration in 50- and 6-µm microchannels. (B and E) A375-SM cells were treated with NSC23766, blebbistatin, or vehicle control, and their migration velocities in channels of different widths were quantified. In B, the images of cells migrating inside the 50- or 6-µm channels at designated time points are shown. White arrowheads show the centroid of the cell body. (C and D) A375-SM cells were plated on a 2D surface (C) or induced to migrate inside a 6-µm channel (D) in the presence of vehicle control (Ctrl), NSC23766 (NSC), or blebbistatin (Bleb), stained with TRITC-conjugated phalloidin, and imaged by confocal microscopy. The boxes at the bottom left corner of each image show enlarged images of stress fibers. In C, the density of stress fibers was measured and graphed as a percentage of total cell spreading area occupied by stress fibers. In D, the dotted lines indicate the PDMS walls of the channel. Each data (A–E) represent means ± SEM of >40 cells from three independent experiments. *, P < 0.005 (B and E) or P < 0.05 (C). (F) A375-SM cells were treated with the α4/paxillin-binding inhibitor, 6B345TTQ, in the presence of NSC23766 or vehicle control. Cell migration velocities in channels of different widths were quantified. Data represent means ± SEM of >30 cells from two independent experiments for each channel width. *, P < 0.005 relative to control. ^§, P < 0.005 relative to 6B345TTQ treatment alone. FN, fibronectin.