pH of organelles of the endosomal-lysosomal compartment. HBMECs were either mock-infected or adsorbed with reovirus T3D at an MOI of 1 PFU/cell, incubated for 4 h, and then incubated for an additional 16 h with FITC- and rhodamine–dextran probes. (A) Phase-contrast image and confocal single optical sections of an infected cell showing compartments labeled with FITC (green, more neutral) and rhodamine (red, more acidic) probes. Arrows indicate yellow lysosome-like organelles that contain both FITC and rhodamine probes adjacent to a VI (asterisk) as an example of pVI organelles included in the pVI quantification. (B) Results are presented as median ± SEM of three independent experiments. The median pH values of endosomal–lysosomal compartments in uninfected and reovirus-infected cells are 6.98 and 6.69, respectively. The pH of organelles adjacent to VIs (pVI) is 6.1. This value differs significantly from the pH of the endosomal–lysosomal compartment in infected cells (***, P < 0.001, unpaired two-tailed Student’s t test). (C) Phase-contrast images and confocal single optical sections of four infected cells showing yellow lysosome-like organelles (arrows) adjacent to VIs (asterisks). The white ellipse surrounds a group of these organelles. Images on the right show the same cells after immunofluorescence with anti-σ1 antibody. Lysosome-like organelles, either with or without virions contained within, have the same color and a pH of ∼6.1. N, nucleus. Scale bars, 3 µm.