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

Figure 5. E. coli O104:H4 OMVs deliver OMV-associated virulence factors intracellularly and cause cytotoxicity via Stx2a.

Figure 5

(a) Cells were incubated for 6 h with OMVs from strains LB226692 or C227-11Φcu or without OMVs (control). Cell lysates were analysed by immunoblot for OMVs (anti-OmpA), Stx2a, ShET1, H4 flagellin, and actin (loading control); signals were visualised with Chemi Doc XRS imager. Sizes of immunoreactive bands are shown on the right side. Crops of representative immunoblots are shown. Full immunoblots are shown in Supplementary Fig. S9. (b–d) Cells were incubated for 6 h with OMVs LB226692 or C227-11Φcu. OMVs were stained with mouse anti-E. coli LPS antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG (green), Stx2a, H4 flagellin (Fla) and ShET1 with their respective antibodies and Cy3-conjugated goat anti-rabbit IgG (red), and nuclei with DRAQ5 (blue). 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 fluorescence images were merged (left panels; colocalised red and green signals appear in yellow) and single fluorescence channels are shown in the right panels. Pictures consisted of one optical section of a z-series with a pinhole of 1 airy unit. Scale bars are 10 μm. (e) Cells were incubated for 72 h with LB226692 or C227-11Φcu OMVs or remained untreated (control); cells were stained with crystal violet, analysed microscopically (Axio Imager.A1) and photographed (AxioCam CCD camera). For LB226692 OMVs photomicrographs of cells displaying ~50% cytotoxicities are shown. Scale bars are 20 μm.