Exosomes containing HIV protein Nef reorganize lipid rafts potentiating inflammatory response in bystander cells
Fig 6
ExNef reorganize lipid rafts and potentiate inflammation via the ABCA1-Cdc42-actin axis.
A—The effect of exNef (0.4 ng/ml) on activation of Cdc42 by bradykinin (BRK, 100 ng/ml). Cdc42 activity was assessed by G-LISA, the assay detects concentration of GTP-bound (active) Cdc42. Mean ± SEM is shown; *p<0.05 versus non-activated cells; #p<0.01 versus activated cells treated with exGFP; (n = 4); B–The effect of exNef (0.4 ng/ml) on the abundance of total Cdc42 (Western blot). C–The effect of exNef (0.4 ng/ml, 48 h) on the abundance of F-actin (Alexa Fluor 488 Phalloidin staining); Scale bars– 10 μm. D–Quantitation of the effect of exNef on the abundance of F-actin. Mean ± SEM is shown; **p<0.01; E–The effect of transfection of cells with with GFP (top row) or Cdc42(Q61L)-GFP (bottom row) before exposure to exNef (0.4 ng/ml, 24 h) on the abundance of rafts (CTB staining, confocal microscopy); Scale bars– 10 μm. F–Quantitation of the effect of transfection of cells with GFP or Cdc42(Q61L)-GFP before exposure to exNef (0.4 ng/ml, 48 h) on the abundance of rafts (CTB staining, confocal microscopy); ***p<0.001 versus GFP-transfected cells; #p<0.05 versus corresponding exGFP-treated cells. G–The effect of transfection of cells with with Cdc42(Q61L)-GFP before exposure to exNef (0.4 ng/ml, 24 h) on the localization of TREM-1; left panel, Cdc42-GFP (transfected cells), middle panel, TREM-1, right panel, merge. Scale bars 10 μm; H–The effect of transfection of cells with with Cdc42(Q61L)-GFP before exposure to exNef (0.4 ng/ml, 24 h) on the localization of TLR4; left panel, Cdc42-GFP (transfected cells), middle panel, TLR4, right panel, merge. Scale bars 10 μm. I—The effect of transfection of cells with with Cdc42(Q61L)-GFP before exposure to exNef (0.4 ng/ml, 48 h) and stimulation with LPS (100 ng/ml, 18 h) on secretion of TNFα. Means ±SEM is shown; **p<0.01 versus exGFP. J—The effect of transfection of cells with Cdc42(Q61L) on the abundance of phosphorylated ERK1/2 in RAW 264.7 macrophages treated with exNef (0.4 ng/ml, 48 h) (Western blot).