Figure 5.

FOXO1 regulates CD11b expression. (A) Neutrophils were isolated from FOXO1-deleted LyzM.Cre⁺FOXO1^L/L and control LyzM.Cre⁻FOXO1^L/L mice and incubated with Porphyromonas gingivalis. (A) RNA was isolated from neutrophils and CD11b mRNA levels were measured by RT-PCR and normalized to ribosomal protein L32. (B) HL-60 neutrophils were transfected with a plasmid expressing FOXO1 or empty vector alone with or without incubation with bacteria. RNA was isolated from neutrophils and CD11b measured by real-time PCR. (C) HL-60 neutrophils were transfected with FOXO1 expression plasmic or vector alone. Immunofluorescence was carried out using a CD11b specific antibody and DAPI counterstain. Intensity was measured to assess protein levels of CD11b. (D) Cells described in panel C were assessed for mean fluorescence intensity (MFI) to quantify CD11b protein levels. (E) FOXO1 interaction with the CD11b promoter examined by chromatin immunoprecipitation (ChIP) assay using primary murine neutrophils from normal mice. The data are representative of two or three independent experiments and expressed as the mean ± SEM from triplicate samples. *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). *Significant difference between neutrophils from transfection of FOXO1 vs. empty vector alone (P < 0.05).