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. 2015 Aug 18;5:13252. doi: 10.1038/srep13252

Figure 4. E. coli O104:H4 OMVs bind to human IECs and are internalised via dynamin-dependent endocytosis.

Figure 4

(a–c) Kinetics of OMV binding and internalisation. Caco-2 (a) HCT-8 (b) and HT-29 cells (c) were incubated with DiO-labelled OMVs from strains LB226692 or C227-11Φcu for 24 h and fluorescence was measured at the times indicated using FACScan flow cytometer before (total cell-associated OMVs) and after trypan blue (TB) quenching (internalised OMVs). The data were analysed using CellQuestTM Pro software, expressed as geometric means of fluorescence intensities from 10,000 cells after subtraction of background fluorescence of cells without OMVs, and are presented as means ± standard deviations from three experiments. (d–g) CLSM of OMV uptake. Caco-2 (d) HCT-8 (e) and HT-29 (f) cells were incubated with OMVs from strains LB226692 or C227-11Φcu for the times indicated. OMVs (green) were detected with anti-E. coli O104 LPS antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG, actin (red) with phalloidin-TRITC and nuclei (blue) with DRAQ5. (g) Control cells incubated for 24 h with OMV buffer instead of OMVs and stained as above. Pictures were taken using a laser-scanning microscope (LSM 510 META microscope, equipped with a Plan-Apochromat 63x/1.4 oil immersion objective). All three images were merged and confocal Z-stack projections are included in panels (d–f). The cross hairs show the position of the xy and yz planes. Scale bars are 10 μm. (h–j) Effects of inhibitors of endocytosis on OMV uptake. LB226692 and C227-11Φcu OMVs labelled with rhodamine isothiocyanate B-R18 were incubated for 6 h with Caco-2 (h), HCT-8 (i) and HT-29 (j) cells which had been pretreated (30 min) with the indicated inhibitors or remained untreated. After solubilisation of cells with Triton-X-100 fluorescence was measured (FLUOstar OPTIMA) and OMV uptake (reflected by the fluorescence intensity) in the presence of each inhibitor was expressed as the percentage of OMV uptake by control, inhibitor-untreated cells (100%). **P < 0.01, and ***P < 0.001 compared to inhibitor-untreated cells (one-way ANOVA). Data are means ± standard deviations from three independent experiments.