FIG. 6.
HES-1 binding to S1 of the CD4 silencer leads to repression of CD4 promoter and enhancer function and endogenous gene expression. (A) Transfection of the pT-based vectors into the D10 TH clone. Transfection of the CMV-HES-1 expression plasmid (H) and of the empty CMV expression plasmid (P) is indicated. Data from each reporter construct set are presented normalized to the results obtained from the transfection with empty plasmid. Data were also normalized for transfection efficiency using Renilla luciferase values as described in Materials and Methods. Total amount of DNA added to all transfections was kept constant with the addition of empty plasmid DNA as needed. Multiple transfection experiments were conducted; presented are averages of data from three independent transfections. (B) Representative two-color flow cytometric plot of D10 cells cotransfected with the CMV-GFP and CMV-HES-1 expression plasmids. For this experiment, D10 TH cells were cotransfected with (top) or without (bottom) the CMV-HES-1 expression plasmid and the CMV-GFP plasmid; GFP-positive cells were gated on and analyzed for surface CD4 and CD3 expression. (C) Percentage of transfected CD4dull SP D10 TH cells plotted against the amount of EF-1α-HES-1 plasmid added to the transfection. Total amounts of DNA added to the transfections was kept constant with the addition of irrelevant plasmid DNA as needed. Multiple experiments were conducted; shown are three representative experiments.