FIG 4.

Knockdown of Stx6 disrupts HCMV trafficking to the TGN in monocytes. Primary human monocytes were treated with siRNA targeting Stx6 or scrambled (Scr) siRNA control and incubated for 48 h for maximal knockdown. (A) An immunoblot was performed on whole cell lysate (representative blot shown). Stx6 band density was measured using LI-COR Image Studio software. Band density was normalized to Scr control to quantify knockdown efficiency (B). The cells were then synchronously infected with HCMV TB40/E UL32-GFP (MOI = 10), then fixed at 15 mpi, 2 hpi, and 4 hpi. Cells were then permeabilized and stained for markers of early endosome (EEA1), the TGN (TGN46), late endosome (M6PR), recycling endosome (Rab11), and HCMV (anti-GFP). 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. Representative images are shown (C). Yellow arrows indicate co-localization. Viral particle distance to the nearest edge of the target organelle was quantified from at least 10 cells (D–G) using the microscope companion analysis software. Student’s t-tests were used to assess statistical significance. **, P < 0.01; ***, P < 0.001. A–E: N = 3; F–G: N = 2.