Figure 2. Silencing of Rab27a impairs... | Rockefeller University Press

Figure 2.

$Figure 2. Silencing of Rab27a impairs the recruitment of MVEs to the site of HIV-1 assembly and Pr55Gag association with the PM in Jurkat cells. (A) LSCM of live control and Rab27a-silenced cells stably expressing CD63-GFP and stained with LysoTracker. (B) FACS plots showing endogenous cell surface CD63 levels in control and Rab27a-silenced cells. Isotype control is shown (gray line). The data shown are from a single representative experiment out of six repeats. For the experiment shown, 10,000 cells from each condition were analyzed. (C) Mean fluorescence intensity (MFI) of cell surface CD63 staining in control and Rab27a-silenced cells (n = 6). (D) Immunofluorescence LSCM images of endogenous CD63 and Gag in control and Rab27a-silenced infected Jurkat cells at day 12 p.i. To better visualize Gag cytosolic distribution in Rab27a-silenced cells, the laser power was increased for the acquisition of the green channel. (E) Quantitation of PM versus cytosolic distribution of Gag and of CD63 forming a PM cap versus its intracellular location in 100 cells of each condition. Data are expressed as percentages of cells in each category. (F) LSCM of control and Rab27a-silenced Jurkat cells. Cell surface CD81 was stained in nonpermeabilized cells, and, subsequently, intracellular Gag was stained. (G) Quantitation of PM versus cytosolic distribution of Gag was evaluated in 100 cells of each condition. Data are expressed as percentages of cells in each category. (H) Immunoblots of cell fractions to analyze the distribution of Pr55Gag and p24. The purity of the fractions was assessed by detection of the cytosolic protein GAPDH and the membrane-associated protein calnexin. M, membrane fraction; C, cytosolic fraction. One representative experiment out of three is shown. (I) Ratio of cytosol/membrane distribution of p55, corresponding to three independent experiments. In A, D, and F, transmitted light channel is shown on the right. The quantifications shown in E and G were performed by blinded observers on a per cell basis. KD, knockdown. **, P < 0.01; ***, P < 0.001. Bars, 2 µm.$

Silencing of Rab27a impairs the recruitment of MVEs to the site of HIV-1 assembly and Pr55^Gag association with the PM in Jurkat cells. (A) LSCM of live control and Rab27a-silenced cells stably expressing CD63-GFP and stained with LysoTracker. (B) FACS plots showing endogenous cell surface CD63 levels in control and Rab27a-silenced cells. Isotype control is shown (gray line). The data shown are from a single representative experiment out of six repeats. For the experiment shown, 10,000 cells from each condition were analyzed. (C) Mean fluorescence intensity (MFI) of cell surface CD63 staining in control and Rab27a-silenced cells (n = 6). (D) Immunofluorescence LSCM images of endogenous CD63 and Gag in control and Rab27a-silenced infected Jurkat cells at day 12 p.i. To better visualize Gag cytosolic distribution in Rab27a-silenced cells, the laser power was increased for the acquisition of the green channel. (E) Quantitation of PM versus cytosolic distribution of Gag and of CD63 forming a PM cap versus its intracellular location in 100 cells of each condition. Data are expressed as percentages of cells in each category. (F) LSCM of control and Rab27a-silenced Jurkat cells. Cell surface CD81 was stained in nonpermeabilized cells, and, subsequently, intracellular Gag was stained. (G) Quantitation of PM versus cytosolic distribution of Gag was evaluated in 100 cells of each condition. Data are expressed as percentages of cells in each category. (H) Immunoblots of cell fractions to analyze the distribution of Pr55^Gag and p24. The purity of the fractions was assessed by detection of the cytosolic protein GAPDH and the membrane-associated protein calnexin. M, membrane fraction; C, cytosolic fraction. One representative experiment out of three is shown. (I) Ratio of cytosol/membrane distribution of p55, corresponding to three independent experiments. In A, D, and F, transmitted light channel is shown on the right. The quantifications shown in E and G were performed by blinded observers on a per cell basis. KD, knockdown. **, P < 0.01; ***, P < 0.001. Bars, 2 µm.