E. coli OMVs are internalised by and localise to the perinuclear regions of Caco-2 cells. Fluorescent DiO–labelled OMVs (green), were added to Caco-2 cells for 4 hours before the cells were stained for actin (red) and nuclei (blue). Image stacks were taken 0.28 μm apart. The top images represent a maximum intensity z-projection constructed in ImageJ whereas the bottom images are insets of those above with their x-z and y-z orthogonal projections to show that OMVs are internalised by Caco-2 cells. Images are from a single representative experiment. Original magnification = 40×, NA1.3 objective used. Note that the figure for O157:H7 OMV treated Caco-2 cells shows an enlarged, intoxicated nucleus.