Fig. 4. Increasing doses of UV increase the activation of p38, but not ERK kinase, and p38 activation is necessary for UV-induced NETosis.

a Human neutrophils were collected 1 h after activation with PMA (25 nM), A23 (4 µM) or increasing doses of UV for western blot analysis. Immunoblots show that increase in UV dose increases p38 activation (determined by detecting phosphorylated p38 kinase). Densitometry analysis confirms the significant increase in p38 activation following UV treatment (n = 3). b Immunoblots show that ERK is not activated (determined by detecting phosphorylated ERK kinase) during UV irradiation. Densitometry analysis confirm the lack of ERK activation following UV treatment (n = 3). c, d Cells were incubated with p38 inhibitor SB203580 (20 µM) for 1 h prior to UV (1.92 J/cm²) treatment, and analysed with SYTOX Green assay. d Neutrophils were incubated with SB203580 (20 µM) for 1 h and then treated with UV (1.92 J/cm²) and incubated for 240 min. Cells were stained for DNA (DAPI, blue). Fluorescence imaging shows that SB203580 inhibits UV-induced NETosis (scale bar, 10 μm). Images are representative of three independent experiments. For all the graphs: error bars represent SEM; A23, A23187; SB, SB303580; *p < 0.05 for comparing UV treatment alone to UV treatment with inhibitors