p53 Transcriptional Activity by Dual-Luciferase Reporter Assay

The two cell lines were seeded in white 96-well plate (1.5 × 10⁴ cells for MRC-5 or 3.5 × 10⁴ cells for XPA) in triplicate for each condition. One day after plating, a total of 105 ng plasmid DNA [100 ng pGL3-p53RE (p53 responsive element, or RE) containing the Firefly luciferase reporter, and 5 ng of pShuttle, containing the Renilla luciferase reporter] (Yu et al., 2007) were transfected using the transfection agent Lipofectamine 3000 (Life Technologies) according to the manufacturer protocol. After 24 h of transfection, cells were then irradiated (18 J/m² UVC) and maintained in culture for a further 18 h when the Dual-Glo kit Luciferase Assay System (Promega) was used to measure the activity of the reporter genes. The reporter gene luciferase expression/activity was read using the luminometer Glomax-Multi Detection System (Promega). The data were then processed, where the Firefly luciferase signal was normalized for the Renilla luciferase signal, and this ratio was assumed as the percentage of levels of p53 transcriptional activity.