Figure 4

Epoxomicin restores CycT1-U7 and Tat expression. A. HA-tagged wt and mutant CycT1 and myc-tagged Tat proteins were co-expressed in HeLa/HR-Luc cells. Twenty-four hours after transfection, cells were untreated or treated with 25 μM Epoxomicin for 3 hours. HA-CycT1 proteins were visualized with mouse anti-HA antibody and Cy2-conjugated donkey anti-mouse IgG. Myc-Tat proteins were seen with Texas Red-conjugated anti-myc antibody. Nuclei were stained with Hoechst. B. The inhibitory effect by CycT1-U7 was diminished by Epoxomicin. HeLa/HR-Luc cells were transfected with CycT1-U7 expression plasmids (0.5 μg) or empty plasmids (0.5 μg) in the presence of Tat. Cells were treated with DMSO (-) or 25 μM Epoxomicin for 6 hours and 18 hours as indicated, and the Luc activities were measured. The results were presented as relative luciferase values obtained with CycT1-U7 divided by the values with the empty vector at each time point. C. Increasing amounts of Tat were transfected in Hela/pHR-Luc cells stably expressing CycT1-U7 proteins. 24 hours after transfection, cells were untreated (open circles) or incubated (closed circles) with Epoxomicin (25 μM) for 6 hours prior to luciferase assay. Data are presented as fold activation relative to the value obtained with untreated cells. Error bars represent the standard deviation of triplicate measurements. Data are representative of three independent assays.