EXOC2 depletion abrogates Nef-mediated enhancement of nanotube formation. A) Lysates of Jurkat E6-1 cells transduced with vector virions bearing pLKO.1-shRNA followed by pHAGE-Nef-IRES-ZsGreen encoding 5C (wild-type) or empty pHAGE were harvested 32 h post-pHAGE transduction and immunoblotted to detect endogenous EXOC2, HA-tagged Nef, and β-tubulin. Samples were treated with an shRNA targeting EXOC2 (right lane, EXOC2) or a control shRNA that does not target the human transcriptome (left lane, Control). B -F) Jurkat cells transduced as described above were stimulated with 1 ug/mL PHA-P for 1 h at 24 h post-pHAGE transduction, incubated on fibronectin-coated coverslips for 5 h, fixed at 30 h post-pHAGE transduction and phalloidin-stained prior to visualization by confocal fluorescence microscopy. B) Percentages of ZsGreen-positive Jurkat cells that formed nanotubes and nanotube-like structures in populations treated with shEXOC2 (right) or shControl (left) are shown. Differences betweenNef-expressing samples treated with control versus EXOC2 shRNAs were significant, as were differences between control shRNA-treated cells with versus without Nef expression (“*” denotes p < 0.05, Mann–Whitney U). The percentage of Nef-expressing cells that formed nanotubes and nanotube-like structures was calculated by manually counting the number of connected cells and dividing the result by the total number of ZsGreen-positive cells for a given sample. The total numbers of ZsGreen-positive cells counted in 30 fields at 40X magnification are detailed in the Methods. Data represent percentages of ZsGreen-positive cells connected by nanotubes, and are reported as mean ± SEM. C-F) Selected fields that contain nanotubes (C and D) and representative confocal fluorescence images (E and F) from the samples quantified in B are shown. Actin-containing nanotubes and nanotube-like structures were visualized via Alexa Fluor 555-conjugated phalloidin; pHAGE-transduced cells were detected via ZsGreen fluorescence. Scale bars, 5 μm.