FIG 7.

Inhibition of PSaV trafficking by blockade of PI3K/Akt and MEK/ERK signaling pathways. (A) LLC-PK cells were incubated with Alexa Fluor 594 (AF594)-labeled PSaV particles (approximately 415 particles per cell) for the indicated times at 37°C in the presence of 200 μM GCDCA. The cells were then fixed, permeabilized, and further incubated with a monoclonal antibody against the early endosome marker EEA1 or the late endosome marker LAMP2. After incubation with a FITC-conjugated anti-mouse IgG antibody, the cells were processed for confocal microscopy to determine the colocalization of AF594-labeled PSaV particles with the early endosome marker EEA1 or the late endosome marker LAMP2. The boxed areas are magnified and shown under each panel. (B and C) LLC-PK cells were pretreated with or without wortmannin (PI3K inhibitor) or U0126 (MEK inhibitor) for 1 h at 37°C and then infected with AF594-labeled PSaV particles (approximately 415 particles per cell) in the presence (B) or absence (C) of 200 μM GCDCA for 3 h. After fixation and permeabilization, the cells were incubated with a monoclonal antibody against EEA1 or LAMP2 and then with a FITC-conjugated secondary antibody to visualize colocalization of AF594-labeled PSaV particles with EEA1 or LAMP2. The boxed areas are magnified and shown under each panel. All experiments were performed in triplicate, and a representative set of results is shown. Bars, 10 μm (A) and 20 μm (B and C). (D and E) Quantification of AF595-labeled PSaV particles colocalized with the early endosome marker EEA1 (D) and the late endosome marker LAMP2 (E) was performed using 10 confocal microscopy images of cells treated under the conditions described above by use of the ImageJ program. Quantification of signals was made with a threshold of 0.03 to 1.3 µm² as described in Materials and Methods.