MLCK controls neutrophil adhesion and integrin activation. (A) dHL-60 cells pretreated with (25μM, 30 minutes) or without ML-7 were not stimulated or stimulated in suspension by 1μM fMLP and allowed to adhere to FN-coated surface for 30 minutes, after which the degree of cell adhesion was assessed. Values were normalized to adhesion in control cells (100%) with fMLP and are mean plus or minus SEM (n = 4). *Results significantly different from control (P < .001). (B) Assessment of adhesion area in leading edge and cell body in control and ML-7–treated cells transfected with EGFP-α5-integrin and exposed to a point source of 10μM fMLP. Cell images were from supplemental Figure 13. Left panel: The time point at which leading edge protrusion of individual cells was maximal was selected for analysis. Leading edge (denoted as “L”) in TIRF image was demarcated by the corresponding DIC image, and the rest of the cell was defined as cell body (denoted as “C”). Right panel: Fluorescence intensities of the leading edge and the cell body in the TIRF images of control and ML-7–treated cells were determined with ImageJ software 1.43, and the resulting values were used to quantify cell attachment of the both leading edge and the cell body. The plot shows the relative values in each region compared with control (100%) in the presence of fMLP stimulation. Values are mean plus or minus SEM (n = 13 for control, 11 for cells treated with ML-7). *Results significantly different from control (P < .0001). (C-D) Top panel: Localization of activated α5β1-integrins in polarized neutrophils pretreated with or without ML-7 (25μM, 30 minutes). dHL-60 cells pretreated with or without ML-7 were plated on FN-coated coverslips and stimulated for 3 minutes by 1μM fMLP. After washing, cells were fixed with 3.7% paraformaldehyde, incubated with GST-FN III_9-11 (50 μg/mL) for 15 minutes at 37°C, and stained with an anti-GST antibody and anti–α5-integrin antibody. Fluorescent and phase-contrast images were collected using confocal fluorescence microscopy. The images of a representative cell for each condition are shown (n = 20 for control and n = 17 for ML-7 treatment). Arrows indicate the leading edge. White line indicates the path along which line profile was obtained. The weak signals for GST-FN III_9-11 binding may be attributed to the relative low levels of activated α5β1-integrin in the cells. Scale bar represents 10 μm. Bottom panel: Line profiles of GST-FN III_9-11 and α5-integrin fluorescence in cells shown in the top panel. The graphs plot fluorescence intensity of each protein (y-axis; in arbitrary units) versus distance (x-axis in pixels) along the white line on the phase-contrast image of the cells. (E-F) Levels of activated α5β1-integrins in cells with or without ML-7 treatment. dHL-60 cells pretreated with or without ML-7 were stimulated by a uniform concentration of fMLP (1μM) in either suspension (E) or adhesion conditions (F). A typical blot is shown on the left. (Right panel) Quantification of blots from 4 separate experiments. The y-axis represents relative intensities (measured with ImageJ software 1.43) with values normalized to the signal (1) detected in the control cells without ML-7 treatment. Each bar represents the mean plus or minus SEM (n = 4). Results significantly different from those of cells without fMLP stimulation: *P < .0001, **P < .001.