Images show colocalization between Rab27a and gD. Colocalization between Rab27a and GHSV-UL46 appears yellow;

between Rab27a and gD, magenta; between GHSV-UL46 and gD, cyan; colocalization between Rab27a, GHSV-UL46 and gD appears white. (DIC: Differential Interference Contrast). Figure 6 Colocalization between Rab27a and viral glycoproteins in the TGN. HOG cells cultured in DM and infected at a m.o.i. of 1 with wild-type HSV-1 were fixed and processed for confocal triple-label indirect immunofluorescence analysis with polyclonal anti-Rab27a and anti-TGN-46 antibodies. Low panels, corresponding to confocal slices of 0.8 μm, are enlargements of the squares shown in upper panels, which correspond to the projection of the planes obtained by confocal microscopy. Colocalization between gD and the TGN appears yellow; between Rab27a ARRY-438162 and the TGN, magenta; between Rab27a and gD, cyan. Arrow VS-4718 in vitro points to colocalization of Rab27a with gD in the TGN. (DIC: Differential Interference Contrast). Effect of Rab27a depletion in HSV-1 infection Further analysis of the role of Rab27a during HSV-1 infection,

was carried out by shRNA knockdown. To generate stably silenced cell lines, HOG cultures were transfected with two plasmids expressing Rab27a shRNAs. One of them, named shRNA-313, induced an selleck products efficient knockdown of Rab27a while, in comparison, a second one, shRNA-735, elicited a weaker effect (Figure 7A and 7B). Figure 7 Effect of Rab27a depletion on HSV-1 infection. HOG cells mock-transfected or transfected with Rab27a-silencing shRNA-313 or shRNA-735, and shRNA non target control, were fixed and processed for confocal immunofluorescence analysis with polyclonal anti-Rab27a antibody. As images show, shRNA-313 Loperamide induced an efficient knockdown of Rab27a while shRNA-735 produced

a weaker effect (A). Equal number of cells were subjected to SDS–PAGE and analyzed by immunoblotting with anti-HSV-1 and anti-GFP antibodies. In Rab27a-depleted cells, a significant decrease in viral-associated GFP signal can be observed (B). Plaque assay shows a drastic reduction in plaque size and a decrease in the viral production determined by the number of plaque forming units (p.f.u.) per ml in silenced shRNA-313 cells compared to control cells (C). Silenced cells and controls infected at a m.o.i. of 1 with K26GFP were processed for flow cytometry, analyzing fluorescence of GFP (D). Percentage (%) of max designates the number of cells relative to the maximum fraction. For each fluorescence intensity within positive cells, the percentage of silenced cells corresponding to shRNA-313 and 735 is considerably lower than controls. Data are representative of 3 independent experiments. E. Rab27a-depleted cells and controls were infected at a m.o.i. of 0.5 with HSV-1. 18 h p.i., viral titers were determined by TCID50. Virus yield was significantly reduced in shRNA-313 silenced cells.