Tiam2 knockdown reproduces the ATF3^−/−neutrophil phenotype in WT neutrophils. Tiam2 knockdown reproduces the ATF3^−/− neutrophil phenotype in WT neutrophils. (A) TIAM2 shRNA knockdown with sequence 1 (sh1) or 2 (sh2) results in a significant reduction in TIAM2 protein expression by western blot in WT neutrophils compared with scrambled shRNA knockdown control. (B-D) Neutrophils transduced with scr (white), sh1 (black), or sh2 (gray) shRNA were placed in a Zigmond slide with chemoattractant gradient (10 nM fMLP) established to the right. Images were captured using a Zeiss Axiovert 200 microscope at 10×/0.3 NA objective, equipped with an ORCA-ER C4742-95 camera driven by Openlab, version 5.5.0 software. (B) Individual paths were traced using Image J software. Cells generating (C) productive (>20 μm) movement and (D) net distance traveled in microns were quantified. (E-F) Transduced neutrophils as in C were allowed to adhere to uncoated glass slides and then stimulated with 10 μM fMLP. Focal complexes and contacts were determined by vinculin-Alexa-fluor 488 stain as in Figure 6. 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. Representative images in E and focal complex quantification in F. Results are from independent experiments: (C) N = 2 to 4 independent acquisitions, (D) N = 13 to 22 cells counted/genotype, and (E-F) N = 38 to 60 cells analyzed/genotype. Statistics are 1-way ANOVA with asterisks indicating differences compared with scr control using Dunnett’s multiple comparison post-test. Data are mean ± SEM. *P < .05, ***P < .001.