Journal: PLoS ONE

Article Title: Involvement of Microtubular Network and Its Motors in Productive Endocytic Trafficking of Mouse Polyomavirus

doi: 10.1371/journal.pone.0096922

Figure Lengend Snippet: Non-transfected 3T6 cells (A and E) or cells transiently expressing EGFP-tagged marker of interest (B–D) were infected with MPyV (MOI of 5×10 2 virus particles per cell) and fixed 5 h p.i. Cells were immunostained for MPyV VP1 capsid protein (red) and for a second marker of interest (caveolin-1, BiP) if not fused with EGFP (green). Confocal sections of cells with enlarged details are shown. Arrowheads point to selected MPyV virions co-localized with the marker of interest. Arrows point to selected MPyV that did not co-localize with the marker of interest. Bars, 10 µm. (F) Quantification of co-localization of MPyV virions with indicated markers at 5 h p.i. in non-treated cells (control) or cells pre-treated (1 h) and infected in the presence of nocodazole (Noc) or latrunculin A (LatA). (G) Quantification of co-localization of MPyV virions with EGFP-Rab5 at 1.5 h p.i., in control, Noc- or LatA-treated cells. The percentage of co-localizing virions was calculated from images such as those showed in A–E. During the experiment, more than 600 virions in at least 10 different cells were evaluated for each sample. Data in the graph represent mean values ± s.d. from three independent experiments.

Article Snippet: Ultrathin cryosections were transferred in a drop of 2.3M sucrose, 2% methylcellulose onto formwar-carbon-coated nickel grids and immunolabeled with rabbit polyclonal antibody against caveolin-1 (BD Transduction Laboratories, BD Biosciences), followed by incubation with goat anti-rabbit antibody conjugated to 10 nm gold particles (British Biocell Int.).

Techniques: Transfection, Expressing, Marker, Infection

Journal: PLoS ONE

Article Title: Involvement of Microtubular Network and Its Motors in Productive Endocytic Trafficking of Mouse Polyomavirus

doi: 10.1371/journal.pone.0096922

Figure Lengend Snippet: 3T6 cells were pre-treated (1 h) with nocodazole, infected with MPyV in the presence of the drug and fixed 5 h p.i. (A) Cells immunostained for MPyV VP1 (red) and caveolin-1 (green). (B) Cells expressing EGFP-fused Rab7 GTPase (green) immunostained for VP1 protein (red). Confocal sections of cells are shown. Arrowheads point to selected MPyV virions co-localizing with indicated marker. Bars, 10 µm. (C and D) Immunolabeling of thawed cryosections of cells with anti-caveolin-1 antibody, followed by immunolabeling with secondary antibody conjugated with 10 nm gold particles (seen as darkly stained dots). Arrowheads point to selected virions. Empty arrowheads point to flask-shape “empty” caveolar structures. Bars, 100 nm. Pm, plasma membrane; MVBs, multivesicular bodies.

Article Snippet: Ultrathin cryosections were transferred in a drop of 2.3M sucrose, 2% methylcellulose onto formwar-carbon-coated nickel grids and immunolabeled with rabbit polyclonal antibody against caveolin-1 (BD Transduction Laboratories, BD Biosciences), followed by incubation with goat anti-rabbit antibody conjugated to 10 nm gold particles (British Biocell Int.).

Techniques: Infection, Expressing, Marker, Immunolabeling, Staining

Journal: PLoS ONE

Article Title: Involvement of Microtubular Network and Its Motors in Productive Endocytic Trafficking of Mouse Polyomavirus

doi: 10.1371/journal.pone.0096922

Figure Lengend Snippet: Thawed cryosections were immunolabeled with anti-caveolin-1 antibody followed by incubation with secondary antibody conjugated with 10 nm gold particles. Arrowheads point to selected virions. Bars, 100 nm. Pm, plasma membrane; MVBs, multivesicular bodies; MLB, multilamellar body; Nu, nucleus.

Article Snippet: Ultrathin cryosections were transferred in a drop of 2.3M sucrose, 2% methylcellulose onto formwar-carbon-coated nickel grids and immunolabeled with rabbit polyclonal antibody against caveolin-1 (BD Transduction Laboratories, BD Biosciences), followed by incubation with goat anti-rabbit antibody conjugated to 10 nm gold particles (British Biocell Int.).

Techniques: Immunolabeling, Incubation

Journal: PLoS ONE

Article Title: Involvement of Microtubular Network and Its Motors in Productive Endocytic Trafficking of Mouse Polyomavirus

doi: 10.1371/journal.pone.0096922

Figure Lengend Snippet: (A) 3T6 cells were pre-treated in culture medium alone or in medium supplemented with nocodazole for 1 h at 37°C and incubated with MPyV (MOI of 5×10 2 virus particles/cell) for 15 or 90 min at 37°C, also in the presence or absence of the drug. After that, immunofluorescence analysis was performed using an anti-MPyV VP1 antibody added to live cells, followed by fixation. Confocal sections of representative cells are shown. Bars, 10 µm. (B) Immunolabeling of thawed cryosections of cells pre-treated and infected with MPyV in the presence of nocodazole. Cells were immunolabeled with anti-caveolin-1 antibody, followed by immunolabeling with secondary antibody conjugated with 10 nm gold particles (seen as darkly stained dots). Arrowheads point to selected virions. Empty arrowhead points to caveolar invagination. Asterisk indicates virion internalizing to an invagination lacking caveolin-1. Arrow points to virion internalizing via a region at the plasma membrane enriched for caveolin-1. Pm, plasma membrane. Bars, 50 nm. (C) 3T6 cells were pre-treated (1 h) with nocodazole and infected with MPyV. The drug was washed out at 7 h p.i. and cells were further incubated until 24 h p.i. (middle bar) or for additional 24 h after washing (right bar). As a control, cells were infected in the absence of the drug and fixed 24 h p.i. Cells were immunostained for MPyV LT antigen and the efficiency of infection was determined by the levels (%) of LT antigen-positive cells, normalized to that in control. During the experiment, at least 500 cells of each sample were counted. Data in the graph represent mean values ± s.d. from three independent experiments. Immunofluorescent staining of microtubules (anti-α-tubulin antibody; panel on the right) shows the morphology of microtubular network at the time of washing (7 h p.i.) in control or nocodazole-treated cells. Bars, 10 µm.

Article Snippet: Ultrathin cryosections were transferred in a drop of 2.3M sucrose, 2% methylcellulose onto formwar-carbon-coated nickel grids and immunolabeled with rabbit polyclonal antibody against caveolin-1 (BD Transduction Laboratories, BD Biosciences), followed by incubation with goat anti-rabbit antibody conjugated to 10 nm gold particles (British Biocell Int.).

Techniques: Incubation, Immunofluorescence, Immunolabeling, Infection, Staining