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. 2019 Jul 25;15(7):e1007907. doi: 10.1371/journal.ppat.1007907

Fig 5. ExNef potentiate inflammatory signalling cascade via re-localization of TLR4 and TREM-1 to lipid rafts.

Fig 5

A–The effect of exNef (0.4 ng/ml, 48h) on the abundance of lipid rafts and re-localization of TLR4 and TREM-1 to the plasma membrane in RAW264.7 macrophages. Left column, CTB staining; second column, anti-TREM-1 staining; third column anti-TLR4 staining; fourth column–merge TREM-1/rafts, right column–merge TLR4/rafts. Scale bars 10 μm; B–Quantitation of the effect of exNef (0.4 ng/ml, 48 h) on the abundance of lipid rafts, TREM-1 and TRL4 at the plasma membrane. Mean ± SEM are shown; **p<0.01, ***p<0.001 versus exGFP; C–Bio-assay for inflammatory cytokines secreted by macrophages treated with exNef (0.4 ng/ml, 48 h) after stimulation with LPS (100 ng/ml of LPS for 18 h). Luminescence produced by SVEG/VCAM endothelial cells (stably transfected with luciferase under VCAM-1 promoter) is shown (see Methods for details). **p<0.001. D–The effect of exNef on TNFα secretion by human monocyte derived macrophages with or without stimulation with 1 ng/ml of LPS for 24 h; *p<0.05. E–The effect of exNef on IL-6 secretion by human monocyte derived macrophages with or without stimulation with 1 ng/ml of LPS for 24 h; *p<0.05. F–The effect of exNef (0.4 ng/ml, 48 h) on the abundance of total and phosphorylated ERK1/2 in RAW 264.7 macrophages (Western blot); G–Ratio of phosphorylated to total ERK1/2 (Western blots, n = 3, *p<0.05); H- Release of Il-1β from BMDM pre-treated with exNef (0.4 ng/ml, 48 h) and treated with LPS (100 ng/ml, 4 h)) and Nigericin (5μM, 3 h). Control cells were treated with exGFP; n = 4, ***p<0.001. I–The effect of exNef (0.4 ng/ml, 48 h) on the abundance of pro-caspase-1 in the BMDM cell lysate and of the cleaved p10 form immunoprecipitated from the cell culture medium (Western blot); J–The effect of exNef (0.4 ng/ml, 48 h) on apoptosis and necrosis in RAW 264.7 macrophages (TUNEL assay).