Figure 3.

FOXO1 deletion impairs neutrophil bacterial phagocytosis. (A) Carboxyfluorescein succinimidyl ester (CFSE)-labeled bacteria were incubated with human HL-60 neutrophils transfected with scrambled or FOXO1siRNA. Deconvolution fluorescent microscopic images were taken after the neutrophils were stained with Alexa 647-labeled wheat germ agglutinin (WGA) to delineate the cell surface (red) and internalized bacteria (green). Internalized bacteria were considered as those within WGA-labeled plasma membranes. (B) Internalized bacteria expressed as the number of bacteria internalized per cell. (C) The percent neutrophils with phagocytosed bacteria. (D) The percent neutrophils with associated bacteria. (E) FOXO1-deleted LyzM.Cre⁺FOXO1^L/L and control LyzM.Cre⁻FOXO1^L/L mice were inoculated with Porphyromonas gingivalis in vivo. Live bacterial CFUs were measured. (F) Neutrophils from FOXO1-deleted LyzM.Cre⁺FOXO1^L/L mice and littermate control LyzM.Cre⁻FOXO1^L/L mice were isolated and incubated with P. gingivalis in vitro. Viable bacterial CFUs were counted. (G) RNA was isolated from neutrophils and FOXO1 was measured by real-time PCR. (H) Neutrophils transfected with the siRNA were analyzed by Western blots using a antibody specific for FOXO1. Actin was assessed as a loading control. The data are representative of two or three independent experiments of mean ± SEM from triplicate samples. *Significant difference between human neutrophils from transfection of FOXO1 siRNA vs. scramble siRNA (P < 0.05). *Significant difference between neutrophils from FOXO1-deleted LyzM.Cre⁺FOXO1^L/L mice and littermate control LyzM.Cre⁻FOXO1^L/L mice (P < 0.05). SCR, scrambled siRNA; UT, untransfected control.