F protein activates ERK and p38 MAPK to induce NET formation.

(A,B) Neutrophils (2 x 106/mL) were pretreated with PD98059 (30 μM) or SB203580 (10 μM) for 1 h and stimulated with F protein (1 μg/mL) for 3 h at 37°C with 5% CO2. NETs were quantified in culture supernatants using Quant-iT dsDNA HS kit. Data are representative of 3 separate experiments performed in triplicates and represent mean ± SEM. ***p<0.001 when compared to negative control (NCtrl); #p<0.001 when compared to F protein-treated cells. (C,D) Neutrophils (1 x 106/mL) were stimulated with F protein (1 μg/mL) for 5 min at 37°C with 5% CO2 and stained for phosphorylated proteins (ERK 1/2 and p38 MAPK), according to Materials and Methods. Proteins phosphorylation was analyzed by flow cytometry using FACSCanto II flow cytometer. Neutrophils gate was based on FSC x SSC distribution. Phosphorylation of protein pathways are presented as fold increase relative to unstimulated neutrophils (NCtrl). Data are representative of 2 separate experiments with similar results.