Figure 2

A. E2F-1 expression up-regulates the activity of the PUMA promoter in SK-MEL-2 cells. SK-MEL-2 cells were co-transfected with a total of 0.88 μg of DNA, including 0.8 μg of PUMA promoter reporter plasmid and 0.08 μg phRL-CMV plasmid (as an internal control) in a 12-well plate by using Lipofectamine 2000 transfection reagent. After 6 hours of transfection, the cells were infected with Ad-LacZ (as control) and Ad-E2F-1. Luciferase activity was examined 16 hours later as described in the manufacturer's protocol using the Dual Luciferase Reporter Assay System (Promega). The bar graphs depict the fold of activation in the luciferase assay after normalization for Renilla luciferase readout values. Each of the experiments is a representation of at least three independent experiments performed in duplicate. B. Transactivation domain of E2F-1 was required for the up-regulation of the PUMA promoter in SK-MEL-2 cells. SK-MEL-2 cells were co-transfected as in Fig. 2A. After 6 hours of transfection, the cells were infected with Ad/T-E2FD (expressing truncated E2F-1) at MOI 100 plus AdTet-off at MOI 5 (using as a helper virus), Ad/T-E2FD at MOI 100 plus AdTet-off at MOI 5, and doxycycline 1 μg/ml or Ad-LacZ at MOI 100. Luciferase activity was examined 16 hours later as in Fig. 2A. The bar graphs depict the fold of activation in the luciferase assay after normalization for Renilla luciferase readout values. Each of the experiments is a representation of at least three independent experiments performed in duplicate.