Effects of Rac1 and myosin II inhibitors on the migration of anti-α4–treated A375-SM, CHO cells and 3T3 fibroblasts in microchannels. (A–C) A375-SM cells treated with an anti–α4 integrin antibody or an isotype control (A), CHO cells (B), or 3T3 fibroblasts (C) were incubated with NSC23766, blebbistatin (Bleb), or the vehicle control (Ctrl) and allowed to migrate through microchannels coated with fibronectin (FN). Cell migration velocities in channels of different widths were quantified. CHO-α4WT cells treated with vehicle control were also included for comparison (B). Data represent means ± SEM of >30 cells from at least three independent experiments for each channel width. *, P < 0.005 relative to control. (D) Cells migrating inside 6-µm channels were 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. The dotted lines indicate the PDMS walls of the channel. (E) Rac1 activity was quantified by measuring the RFP-PAK-PBD red fluorescence intensity for each cell type normalized by FITC intensity from background using dual-color broad-field microscopy. Data represent means ± SEM of ≥85 cells. *, P < 0.05.