Figure 7.
Figure 7. Vpr expression perturbs EB1 and p150Glued localization, leading to a phagosome maturation defect. (A) MDMs were nucleofected at day 5 of differentiation to express HA-Vpr or with the HA plasmid as a control. 5 h later, they were fixed and stained with anti-HA antibodies followed by Cy3-labeled anti–rat IgG (left), DAPI (second lane), and an anti-EB1, followed by Alexa Fluor 488–coupled anti–mouse IgG (middle). Stack of images were acquired and one optical deconvoluted section is shown. Combined images and a 3D reconstitution are shown (right). Bar, 10 µm. (B) Cells were treated as in A and the percentage of EB1 staining localized in the nucleus as detected with the DAPI staining was calculated using Icy software for HA-Vpr–expressing cells and control cells. The dot plot shows the results and means ± SEM from 20 cells of two independent experiments (donors). (C and D) MDMs were nucleofected and treated as in A and B except that p150Glued was stained. Bar, 10 µm. The dot plot shows the results and means ± SEM from 10 cells of one experiment. (E) HeLa cells were transiently transfected to express HA-Vpr or with the HA plasmid as a control. Immunoprecipitation with anti-HA antibodies revealed coimmunoprecipitation of endogenous DHC detected with anti-DHC antibodies. The amounts of total proteins in lysates (1% of total lysates) are shown (right panels). Three independent experiments were performed. (F–K) HeLa cells were transiently transfected to express HA-Vpr or with the HA plasmid as a control, then fixed, and the Duolink proximity ligation in situ assay technology was used with rabbit anti–HA antibodies to detect Vpr combined with mouse mAb anti–EB1 (left panels and I) or mouse anti–p150Glued (middle panels and J), or with mouse anti–HA to detect Vpr combined with rabbit anti–DHC (right panels and K) (H). Negative control was obtained by omitting anti-HA antibody with mouse anti–p150Glued (F) and positive control was with mouse mAb anti–tubulin and rabbit anti–DHC (G). Bars, 10 µm. The number of fluorescent spots was automatically counted using the Icy software SpotDetector function, and the mean number of spots per cell based on nuclei counting in different microscopy fields was plotted (F, G, and I–K). The means ± SEM from three independent experiments are plotted. *, P < 0.05; **, P < 0.005.

Vpr expression perturbs EB1 and p150Glued localization, leading to a phagosome maturation defect. (A) MDMs were nucleofected at day 5 of differentiation to express HA-Vpr or with the HA plasmid as a control. 5 h later, they were fixed and stained with anti-HA antibodies followed by Cy3-labeled anti–rat IgG (left), DAPI (second lane), and an anti-EB1, followed by Alexa Fluor 488–coupled anti–mouse IgG (middle). Stack of images were acquired and one optical deconvoluted section is shown. Combined images and a 3D reconstitution are shown (right). Bar, 10 µm. (B) Cells were treated as in A and the percentage of EB1 staining localized in the nucleus as detected with the DAPI staining was calculated using Icy software for HA-Vpr–expressing cells and control cells. The dot plot shows the results and means ± SEM from 20 cells of two independent experiments (donors). (C and D) MDMs were nucleofected and treated as in A and B except that p150Glued was stained. Bar, 10 µm. The dot plot shows the results and means ± SEM from 10 cells of one experiment. (E) HeLa cells were transiently transfected to express HA-Vpr or with the HA plasmid as a control. Immunoprecipitation with anti-HA antibodies revealed coimmunoprecipitation of endogenous DHC detected with anti-DHC antibodies. The amounts of total proteins in lysates (1% of total lysates) are shown (right panels). Three independent experiments were performed. (F–K) HeLa cells were transiently transfected to express HA-Vpr or with the HA plasmid as a control, then fixed, and the Duolink proximity ligation in situ assay technology was used with rabbit anti–HA antibodies to detect Vpr combined with mouse mAb anti–EB1 (left panels and I) or mouse anti–p150Glued (middle panels and J), or with mouse anti–HA to detect Vpr combined with rabbit anti–DHC (right panels and K) (H). Negative control was obtained by omitting anti-HA antibody with mouse anti–p150Glued (F) and positive control was with mouse mAb anti–tubulin and rabbit anti–DHC (G). Bars, 10 µm. The number of fluorescent spots was automatically counted using the Icy software SpotDetector function, and the mean number of spots per cell based on nuclei counting in different microscopy fields was plotted (F, G, and I–K). The means ± SEM from three independent experiments are plotted. *, P < 0.05; **, P < 0.005.

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