Fig. 3

Macrophage-released EV lipid composition and biogenesis are altered by HG treatments. A Representative SEM images of the surface of macrophages. B Representative TEM images of the membranes of macrophages (scale bar= 500nm). C TEM images of lEVs and sEVs released from untreated macrophages (left). C right, immunogold labelling of macrophage-derived EVs to detect CD63 and CD81 at the EV surface. Red= CD63, 5nm gold particles, yellow= CD81, 15nm gold particles (scale bar=100 nm). D Lipid distribution in lEVs and sEVs from untreated macrophages determined by ¹H-NMR Spectroscopy. DAG: diacylglycerol; TAG: triacylglycerol. E Size distributions of lEVs and sEVs determined by Nanotracking Analyses (NTA) normalized to the total number of detected nanoparticles. F Lipid enrichment determined by¹H-NMR, in lEVs and sEVs released from MG15 and MG30 macrophages compared to untreated macrophages. Values are expressed as log₂ ratios of untreated macrophages. A representative ¹H-NMR spectrum obtained at 600 MHz of CD₃OD/CDCl₃ lipid extracts of lEVs and sEVs is shown in Additional Fig. 4A-B. G Sphingolipid profile of lEVs and sEVs from untreated macrophages, MG15 and MG30 macrophages performed by thin layer chromatography. Representative TLC runs are shown in Additional Fig. 4C. H NTA quantification of lEVs and sEVs, expressed as particle/ml, released from untreated macrophages (UNT), MG15 and MG30 macrophages. Values are means ± SD (n = 3); p values are from student t-test (stimulated vs untreated), (*) p < 0.05, (**) p < 0.01, (***) p< 0.001. MG15=15 mM glucose, MG30=30 mM glucose