FIG 7.

HCMV is retained in EEA1+ vesicles in the absence of Stx6. Primary human monocytes were treated with siRNA targeting Stx6 or a scrambled (Scr) control and incubated for 48 h to ensure maximal knockdown. The cells were then synchronously infected with HCMV TB40/E (MOI = 10). Control and infected monocytes were then fixed at 2 hpi. The cells were then permeabilized and stained with antibodies against GFP (to stain the virus TB40/E UL32-GFP), early endosomes (EEA1) or the TGN (TGN46). High resolution images were acquired on a NIKON N-SIM E Super Resolution microscope system (100× objective) in single slices (z stacks) using the same laser settings. Representative images are shown. Yellow arrows indicate colocalization (A). Viral particle (measured by UL32-GFP) distance to the nearest edge of the target organelle was quantified from at least 10 cells and 20 viral particles using the companion analysis software of the microscope (B). Student’s t-tests were used to assess statistical significance. ***, P < 0.001. N = 3.