Figure 2.

FOXO1 is needed for neutrophil migration and CXCR2 expression. (A) Migration was examined in neutrophils from FOXO1-deleted LyzM.Cre⁺FOXO1^L/L mice or littermate control LyzM.Cre⁻FOXO1^L/L mice in transwell chambers. CXCL1 was added to the bottom chamber and neutrophils that migrated to the bottom chamber were quantified following DAPI staining and fluorescence microscopy. (B) Bacteria were incubated with neutrophils from FOXO1-deleted LyzM.Cre⁺FOXO1^L/L and control LyzM.Cre⁻FOXO1^L/L mice. CXCR2 mRNA levels were measured by RT-PCR and normalized to ribosomal protein L32. (C) HL-60 neutrophils were transfected with FOXO1 or empty vector alone and stimulated with bacteria or vehicle alone. RNA was isolated from neutrophils. CXCR2 expression was measured by real-time PCR. (D) CXCR2 protein levels were assessed by mean fluorescence intensity (MFI). (E) Chromatin immunoprecipitation (ChIP) assays were performed with neutrophils from control LyzM.Cre⁻FOXO1^L/L mice. (F) HL-60 neutrophils were transfected with FOXO1 expression plasmid or empty vector alone and FOXO1 protein levels were measured by immunoblot with a specific antibody. Actin was assessed as a loading control. The data are representative of two or three independent experiments. Data are 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 HL-60 neutrophils from transfection of FOXO1 vs. empty vector alone (P < 0.05).