Reduction of thymic output by HIV-1 Nef requires an intact VGF motif. Flow cytometric analysis of FTOC initiated with retrovirally transduced CD34+ thymic progenitors. (A) Dot plots show eGFP vs. CD4 and eGFP vs. CD3 staining gated on live human cells of Nef transduced cultures, as indicated. Quadrants were set to include 99% of non-transduced cells stained with isotypic controls in lower left quadrant. Numbers in plots indicate mean fluorescent intensity for the marker on the Y-axis (CD4 or CD3) for all eGFP- cells (upper and lower left quadrants) or all eGFP+ cells (upper and lower right quadrants) are indicated. (B) T-cell generation ratio calculated from Nef transduced cultures, as indicated. Bars represent averages of at least 3 independent experiments, error bars indicate standard deviation. Wild-type Nef proteins are shown in dark grey, VGF-AAA mutants in red. Difference compared to control eGFP (white bar) was tested with Mann–Whitney U test, **p < 0.005, ***p < 0.0005.