Inhibition of AP1 recruitment at phagocytic sites in HIV-1 infected cells. (A) Primary human macrophages were infected with HIV-1ADAWT or HIV-1ADAΔNef or noninfected for 8 days. The cells were incubated for 5 minutes with IgG-RBCs, fixed, and stained with AMCA-anti–rabbit IgG to reveal external RBCs. The cells were then permeabilized and labeled with anti-p24 followed by Cy2-anti–goat IgG and with anti-AP1 (γ-adaptin) followed by Cy3-anti–mouse IgG (iv-vi). Merged images show p24 in green and RBCs in red (i-iii) and phase-contrast images are shown (vii-ix). Red arrowheads indicate AP1 enrichment in some forming phagosomes, whereas cyan arrowheads show no enrichment. Bar represents 10 μm. (B) Noninfected cells were treated as described in panel A. (C) The profile of AP1 (γ-adaptin) fluorescence intensities along the lines drawn at the phagocytic site (line 1) and in the cell body (line 2) are shown. (D) The AP1 fluorescence intensity measured in the phagocytic cups were background subtracted and divided by the AP1 fluorescence intensity measured in the cell body after background substraction. This ratio defines the index of recruitment. The means ± SEM of 3 independent experiments, each with 6 noninfected and 6 infected cells, are plotted.
