Fig. 3.

WWOX knockdown in human alveolar epithelial cells results in increased neutrophil chemotaxis. A549 cells grown in culture were transfected with control vs. WWOX-silencing miR-expressing plasmids. A: representative Western blot for WWOX in cell lysates. B and C: transfected A549 cells were grown in the bottom chamber of a Transwell plate containing 3-μm pores. Control IgG vs. neutralizing anti-IL-8 antibody was added to the media. Then, 1 × 10⁵ human neutrophils were placed in the top chamber. Thirty minutes later, neutrophils were pelleted from the media in the bottom chamber and resuspended in a fixed volume, and a cytospin preparation was stained and examined under light microscopy. A representative image is shown here along with a scatter plot depicting the number of neutrophils counted per high-powered field. D–F: concentrations of IL-1β, IL-6, and IL-8 were measured by ELISA in the media supernatant of control vs. WWOX-silenced cells without anti-IL-8 neutralizing antibody or control IgG. Results are shown as means ± SD in n = 3 independent experiments. *P < 0.05, compared with control by Student’s t-test.