ATF3^−/−neutrophils lacking TIAM2 exhibit dysregulated integrin-dependent cytoskeletal organization and adhesive structure regulation. (A) (Upper) WT or (lower) ATF3^−/− neutrophils were allowed to adhere to uncoated glass slides and then stimulated with 10 μM fMLP and stained for polymerized F-actin by phalloidin-rhodamine and focal complexes (arrows) and focal contacts (arrowheads) by antivinculin-Alexa Fluor 488. Representative focal complex/contacts (green), polymerized F-actin (red), 4′6 diamidino-2-phenylindole–stained nuclei (blue), and merged immunofluorescent images are shown. Original magnification, ×630. Representative of 2 (vinculin) or 5 (phalloidin) independent experiments. Fluorescence images were captured at room temperature using a Leica DMI6000 fluorescence microscope at 63×/1.3 NA objective with an ORCA-ER C4742-95 camera driven by Openlab (version 5.5.0) software. Total (B) area and (C) numbers of vinculin-containing focal structures quantified from A for WT (white bars) or ATF3^−/− (black bars) fMLP-stimulated neutrophils. Representative of 2 independent experiments; N = 52 to 83 polarized, nucleus-containing neutrophils/genotype. (D) The total area of F-actin polymerization quantified from A for WT (white bars) or ATF3^−/− (black bars) neutrophils stimulated by fMLP on (left) fibrinogen-coated or (right) uncoated glass slides. Representative of 3 independent experiments; N = 64 to 156 cells counted/genotype. Statistics are unpaired 2-tailed Student t test. Data are mean ± SEM. **P < .01, ***P < .001.