Figure 7

U-937 cell clones containing nonexpressing wild-type (WT) and variant long terminal repeats (LTRs) cannot be induced into driving green fluorescent protein (GFP) expression. U-937 cells stably transfected with the LAI LTR (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). Nonexpressing and expressing cell 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 U-937 cells (solid black line), and treated WT U-937 cells (dashed black line). As there were no nonexpressing U-937 3T5T LTRs containing clones, they are excluded from the non/low-expressing cell clone column. Each clone shown is a representative of the group.