bioimagesUnknownAudrey DumasThe Journal of Cell Biologyhttps://www.ebi.ac.uk/biostudies/studies/S-JCBD-201503124JCB

Primary human macrophages were non-infected, infected with HIV-1YU-2WT or HIV-1YU-2ΔVpr for 8 days. Cells were treated or not (basal) with nocodazole at 10 µM during 1 hour at 37°C. After washing, cells were fixed (time 0) or incubated at 37°C without nocodazole for the indicated times before fixation. Macrophages were then labeled with an anti-p24, followed by Cy2-anti-mouse IgG and a recombinant anti-tubulin, followed by Cy3-anti-human IgG. Images were acquired under a spinning disk microscope. Z stacks images were acquired.

MDM were nucleofected at day 5 of differentiation to express HA-Vpr or with the HA plasmid as a control. Five hours later, they were fixed and stained , anti-HA antibodies followed by Cy3-anti-rat IgG (left panels), DAPI (second lane) and an anti-p150, followed by Alexa Fluor 488-anti-mouse IgG (middle panels). Stack of images were acquired and deconvoluted. Combined images and a three dimensional reconstitution are shown (right panel).

Primary human macrophages were infected with HIV-1ADA WT or mock infected for 8 days. The cells were incubated for different times with IgG-SRBCs at 37°C. Macrophages were fixed, permeabilized and labeled with AMCA-anti-rabbit IgG to detect the total SRBCs, anti-p24 followed by Cy2-anti-goat IgG, anti-LAMP1 followed by Cy3-anti-mouse IgG, and anti-tubuline followed by Cy5-anti-human IgG. Wide field fluorescent images were acquired.

Primary human macrophages were infected with HIV-1ADA WT or mock infected for 8 days. The cells were incubated for different times with IgG-SRBCs at 37°C. Macrophages were fixed, permeabilized and labeled with AMCA-anti-rabbit IgG to detect the total SRBCs, anti-p24 followed by Cy2-anti-goat IgG, anti-LAMP1 followed by Cy3-anti-mouse IgG, and anti-tubuline followed by Cy5-anti-human IgG. Wide field fluorescent images were acquired, non deconvoluted.

MDM were nucleofected at day 5 of differentiation to express HA-Vpr or with the HA plasmid as a control. Five hours later, they were fixed and stained , anti-HA antibodies followed by Cy3-anti-rat IgG (left panels), DAPI (second lane) and an anti-p150, followed by Alexa Fluor 488-anti-mouse IgG (middle panels). Stack of images were acquired and non deconvoluted. Combined images and a three dimensional reconstitution are shown (right panel).

MDM were nucleofected at day 5 of differentiation to express HA-Vpr or with the HA plasmid as a control, incubated for 60 min with IgG-SRBCs at 37°C, then fixed and permeabilized. They were labeled and analyzed as described in Figure 2 (E,F). Z stacks of wide field fluorescent images were acquired, deconvoluted and a Z projection (Image J) is shown.

Primary human macrophages were infected with HIV-1ADA WT or mock infected for 8 days. The cells were incubated for different times with IgG-SRBCs at 37°C. Macrophages were fixed, permeabilized and labeled with AMCA-anti-rabbit IgG to detect the total SRBCs, anti-p24 followed by Cy2-anti-goat IgG, anti-LAMP1 followed by Cy3-anti-mouse IgG, and anti-tubuline followed by Cy5-anti-human IgG. Wide field fluorescent images were acquired, deconvoluted.

Primary human macrophages were non-infected or infected with HIV-1YU-2WT for 8 days. The cells were then fixed and stained with an anti-p24, followed by Alexa Fluor 488-anti-goat IgG and an anti-MICAL-L1, followed by Cy3-anti-rabbit IgG. Z stacks of wide field fluorescent images were acquired.

(F-K) HeLa cells were transiently transfected to express HA-Vpr or with the HA plasmid as a control, then fixed and the Duolink PLA 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).

Primary human macrophages were non-infected (upper panels), infected with HIV-1YU-2WT (middle panels) or HIV-1YU-2ΔVpr (lower panels) for 8 days. The cells were then fixed and stained with an anti-p24, followed by Cy2-anti-goat IgG (not shown) and an anti-p150Glued, followed by Cy3-anti-mouse IgG. Stack of images were acquired.

Primary human macrophages were non-infected or infected with HIV-1YU-2WT for 8 days. The cells were then fixed and stained with an anti-p24, followed by Alexa Fluor 488-anti-goat IgG and an anti-EHD3, followed by Cy3-anti-mouse IgG. Z stacks of wide field fluorescent images were acquired and the stack was deconvoluted.

Primary human macrophages were non-infected or infected with HIV-1Gag-iGFP for 8 days. Gallery of phase contrast images of non-infected (upper panels, see also Movie S1) and HIV-1Gag-iGFP infected (lower panels, see also Movie S2) cells showing phagosome movement.

Primary human macrophages were non-infected or infected with HIV-1ADAWT for 8 days. Total lysates were subjected to western blotting with anti-phospho-ERK1/2 (A), anti-phospho-p38 (B), anti-phospho-SAPK/JNK (C) and anti-phospho-p65/RelA (D). The chemiluminescent signal was quantified and expressed as related to the non-infected condition, showing basal activation by HIV-infection. (E) Macrophages infected for 8 days were incubated for different times with IgG-SRBCs at 37°C, then analyzed by western blot with anti-phospho ERK1/2 and anti-ERK1/2. Results are expressed as a fold increase related to the basal condition for non-infected or HIV-1 infected cells (F). Means ± SEM of three different experiments are plotted. (G) Primary human macrophages were non-infected, infected with HIV-1ADAWT,

HeLa cells were transiently transfected to express HA-Vpr or with the HA plasmid as a control. Immunoprecipitation with anti-HA antibodies revealed co-immunoprecipiation of endogenous DHC detected with anti-DHC antibodies. The amounts of total proteins in lysates (1% of total lysates) are shown (right panels).

Primary human macrophages were non-infected or infected with HIV-1YU-2WT for 8 days. The cells were then fixed and stained with an anti-p24, followed by Alexa Fluor 488-anti-goat IgG and an anti-EHD3, followed by Cy3-anti-mouse IgG. Z stacks of wide field fluorescent images were acquired.

MDM differentiated for 5 days were treated with control siRNA or siRNA against EB1 for 72h. They were then allowed to phagocytose IgG-opsonized SRBCs for 1h, fixed and stained to detect SRBCs with Alexa Fluor488-anti-rabbit IgG (not shown, red in the merge images, right panels) and LAMP1 with anti-LAMP1 followed by Cy3-anti-mouse IgG (middle panels). SRBCs are also detected with phase contrast (left panels). Z stacks of wide field fluorescent images were acquired, deconvoluted and a Z projection (Image J) is shown

MDM were nucleofected at day 5 of differentiation to express HA-Vpr or with the HA plasmid as a control. Five hours later, they were fixed and stained , anti-HA antibodies followed by Cy3-anti-rat IgG (left panels), DAPI (second lane) and an anti-EB1, followed by Alexa Fluor 488-anti-mouse IgG (middle panels). Stack of images were acquired and non deconvoluted. Combined images and a three dimensional reconstitution are shown (right panel).

MDM were nucleofected at day 5 of differentiation to express HA-Vpr or with the HA plasmid as a control. Five hours later, they were fixed and stained , anti-HA antibodies followed by Cy3-anti-rat IgG (left panels), DAPI (second lane) and an anti-EB1, followed by Alexa Fluor 488-anti-mouse IgG (middle panels). Stack of images were acquired and deconvoluted. Combined images and a three dimensional reconstitution are shown (right panel).

Primary human macrophages were non-infected (upper panels), infected with HIV-1YU-2WT (middle panels) or HIV-1YU-2ΔVpr (lower panels) for 8 days. The cells were then fixed and stained with an anti-p24, followed by Cy2-anti-goat IgG (left panels) and an anti-EB1, followed by Cy3-anti-mouse IgG (middle panels). Stack of images were acquired with a wide field microscope.

Primary macrophages differentiated for 5 days were treated with control siRNA (upper panels) or siRNA against MICAL-L1 (A, lower panels) or siRNA against EHD3 (B, lower panels) for 72h. Cells were then fixed, permeabilized and stained with anti-MICAL-L1 followed by Cy3-F(ab’)2 anti-rabbit IgG (A) or anti-EHD3 followed by Alexa488-F(ab’)2 anti-mouse IgG (B). Stacks of images were acquired on a wide field microscope and a maximum Z projection is shown (left panels). Corresponding phase contrast images are shown (right panels). Bar, 10μm.

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