Figure 3: Tamoxifen-induced NET production is mediated by intracellular ceramide.

(a) Ceramide-induced NET production was visualized by immunostaining neutrophils for myeloperoxidase (blue: DAPI; green: NETs/myeloperoxidase); images representative of three independent experiments are shown (scale bar, 50 μm). (b) Quantification of extracellular DNA/NET production in response to ceramide treatment (n=9). (c) The effect of ceramide (30 μM) on ROS production was determined using DCF-based ROS assays (n=9). (d) Neutrophils were pre-incubated with PKC inhibitors (CC=chelerythrine chloride, Gö83=Gö 6983, ζ-PS=PKCζ pseudosubstrate; 10 μM each; n=9) to determine the involvement of PKC in ceramide-induced NET production. (e) Mass spectrometry analysis of lipid extracts was performed to quantify intracellular accumulation of several ceramide species in response to TAM treatment (10 μM). (f) Intracellular ceramide levels in control and TAM-treated (10 μM) neutrophils were visualized via immunostaining of fixed neutrophils using a ceramide primary antibody (green: ceramide); images representative of three independent experiments are shown, with identical exposure and gain settlings used for control and TAM-treated samples (scale bar, 20 μm). (g) The effect of DL-PDMP, which similar to tamoxifen inhibits glucosylceramide synthase, on NET production was assessed via quantification of extracellular DNA (n=9). (h) Myriocin, which blocks ceramide synthesis by inhibiting serine palmitoyltransferase, concentration dependently inhibits tamoxifen-induced NET production. (i) Summary of the mechanism of tamoxifen-induced NET production. By inhibiting glucosylceramide synthase, tamoxifen treatment leads to an accumulation of neutrophil intracellular ceramide levels, which promotes NET formation via activation of PKCζ. Where applicable, results were analysed by one-way analysis of variance and post hoc Newman Keuls test. *P<0.05, **P<0.01, ***P<0.001 versus control values. ROS, reactive oxygen species.