FIG 5.

Stx-associated trafficking disruption is specific to Stx6. Primary human monocytes were treated with siRNA targeting Stx12 or a scrambled control (Scr) (A). An immunoblot was performed on whole cell lysate and Stx12 band density was measured using LI-COR Image Studio software. Band density was normalized to Scr control to quantify knockdown efficiency. A representative blot is shown (A and B). (C and D) Primary human monocytes were treated with siRNA targeting Stx6, Stx12, or a Scr control. The cells were then synchronously infected with HCMV TB40/E UL32-GFP (MOI = 10). The cells were then fixed at 15 mpi and 2 hpi. The fixed cells were then permeabilized and stained for TGN (TGN46) and HCMV (measured via UL32-GFP). High resolution images were acquired on a Nikon N-SIM E Super Resolution microscope system (100x objective) in single slices (z stacks) using the same laser settings. Viral particle (measured via UL32-GFP) distance to the nearest edge of the TGN was quantified from at least 10 cells and 20 viral particles using the companion analysis software of the microscope. Quantification of images is shown. Student’s t-tests were used to assess statistical significance. *, P < 0.05; **, P < 0.01. (E) A viral entry assay (HCMV TB40/E UL32-GFP; MOI = 1) was performed on Stx6, Stx12, or Scr siRNA-treated primary human monocytes. DNA from these cells was then isolated and qPCR was performed with primers for HCMV UL123 and cellular CRP. (F–G) Total viral particles (measured via UL32-GFP) within infected and Stx6 siRNA-treated monocytes were counted at 15 mpi and 2 hpi using the NIKON N-SIM E Super Resolution microscope system (100× objective) in single slices (z stacks) using the same laser settings. Student’s t-tests were used to assess statistical significance. N = 3.