Figure 6

Nonexpressing TF-1 cell clones containing the 3T5T LAI long terminal repeat (LTR) could be induced into LTR-driven green fluorescent protein (GFP) expression. TF-1 cells stably transfected with the HIV-1 LAI LTR (wild type [WT], 3 T, 5 T, and 3T5T) were serially diluted in order to obtain 1 cell in 1 mL of media (approximately 1 cell in 10 wells of a 96-well plate). Cell clone populations were propagated from the single cell and then were analyzed using flow cytometry for their basal GFP expression. The clonal populations were then designated in one of three categories (nonexpresser, intermediate expresser, and high expresser) based on their geometric mean fluorescence intensity (MFI) and their percent cell positive values (Figure 5A). Non/low-expressing and high-expressing LAI WT and LAI 5 T LTR containing clones were treated with a range of tumor necrosis factor-α (TNF-α) concentrations (20–300 ng/mL). Representative histograms (at a TNF-α concentration of 20 ng/mL) showing levels of GFP expression obtained with the untreated, stably transfected cell clone (solid turquoise line) compared with the treated, stably transfected cell clone (dashed turquoise line), untreated WT TF-1 cells (solid black line), and treated WT TF-1 cells (dashed black line). Each clone shown is a representative of the group.