Fig. 5

SFK and PAD-4 signaling is involved in CR Abs-triggered NETosis in vitro. (A) The picture describes the SFK-induced signaling pathway by which IL-8 is produced/secreted or NETs are formed by neutrophils and the target protein of each inhibitor. Isolated human neutrophils were stimulated with mAb 127 (10 μg/mL) and the RBD protein (10 μg/mL) and cotreated in the presence or absence of the indicated inhibitor at the indicated concentrations for 24 h (vehicle control, DMSO; Compound C, an AMPK inhibitor, 30 μM; dasatinib, a src protein family inhibitor, 50 nM; Ly294002, a PI3K inhibitor, 10 μM; Cl-amidine, a PAD-4 inhibitor, 200 μM). (B) The cell suspensions were spun onto a microscope slide by using a cytocentrifuge, fixed and subjected to immunofluorescence staining with an anti-MPO antibody (green), anti-histone antibody (red) and Hoechst (blue) nuclear stain. The supernatants were collected to measure the (C) IL-8 and (D) MPO levels using ELISA kits. (E) NET quantification as determined by immunofluorescence staining. Nine pictures from each group of experiments were randomly selected and analyzed by ImageJ. Bar: 25 μm. The averages of triplicate cultures ± SD are shown. Statistical significance was calculated using one-way ANOVA and Tukey’s post hoc test; P values were displayed, and for values less than 0.0001, they were represented as ****. Bar: 25 μm