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. 2016 May 6;100(4):801–810. doi: 10.1189/jlb.4A0615-261RR

Figure 3. ERK activation upstream of ROS generation is required for netosis triggered by L. amazonensis.

Figure 3.

Neutrophils were left untreated (Nil) or preincubated with PD98 (30 or 60 µM) or AS60 (10 µM) at 5% CO2, 35°C. Cells were then incubated with promastigotes or PMA for 5 or 15 min and stained for intracellular phosphorylated ERK (pERK). (A) Histogram of representative data and (B and F) normalized MFI. Additionally, neutrophils preincubated with PD98 and/or AS60 were stimulated with (C, D, and G) La (10 neutrophils:1 parasite) or PMA (100 nM), and then NET-DNA in the supernatant was quantified as described earlier. Data represent means ± sem of normalized data relative to stimulated cells. Sec Ab, Secondary antibody. ROS generation by DHR 123 probing (E) was measured from untreated or PD98-pretreated neutrophils and stimulated with the parasite or PMA, which was used as positive control. Data represent mean percentages ± sem of neutrophils in the R2 zone. NET-DNA raw numbers: PMA-stimulated cells, 1529 ± 155 ng/ml; La-stimulated cells, 4175 ± 923 ng/ml. *P < 0.05 compared with stimulated cells; #P < 0.05 compared with Nil.