Figure 7.

FOXO1 regulates TNFα and IL-1β. (A,B) Neutrophils from FOXO1-deleted LyzM.Cre⁺FOXO1^L/L mice and littermate control LyzM.Cre⁻FOXO1^L/L mice were incubated with Porphyromonas gingivalis. RNA was isolated from neutrophils and mRNA levels of TNFα or IL-1β were measured by RT-PCR and normalized to ribosomal protein L32. (C–F) HL-60 neutrophils were transfected with FOXO1 or empty vector alone and stimulated with bacteria or vehicle. (C,D) RNA was isolated from neutrophils and mRNA levels of TNFα and IL-1β measured by real-time PCR. (E,F) Cells were incubated with anti-TNFα or anti-IL-1β antibody (sc-7884, Rabbit Polyclonal IgG 1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight and then with secondary antibody, donkey antirabbit biotinylated antibody (1:200, Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at room temperature in a box with the humidified atmosphere, followed by incubation with streptoavidin-Alexa 546 (S-11225, 1:400, for 1 h, Thermo Fisher Scientific). The mean fluorescence intensity (MFI) was measured to assess protein levels of each. 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 HL-60 neutrophils transfected with FOXO1 expression plasmid vs. empty vector alone for mRNA or protein level (P < 0.05).