H1299 cells were transfected with pRL-CMV (Promega), pGL3 Basic with p21 promoter [39 (link)] or with K14 promoter (a gift from Prof. Dr. Karen Vousden (Francis Crick Institute, London, UK)), and pcDNA3.1(+) as an empty vector control or pcDNA3.Myc plasmids encoding the indicated Myc-tagged p53 or p63 variants. 24 h after transfection, cells were harvested and resuspended in fresh medium. Per sample, 45 μl cell suspension was transferred into four wells each of a 96-well plate to determine the luciferase signal in technical quadruplicates. To prepare input samples for western blot analysis, the residual cells were centrifuged, resuspended in 100 μl 2x SDS-PAGE sample buffer and boiled. The luciferase reporter assay was performed using the Dual-Glo luciferase assay system (#E2940, Promega) according to the manufacturers’ recommendation. Luminescence signals were measured using a Spark multimode or a GENios Pro microplate reader (Tecan). The ratio of the Firefly to Renilla luciferase signal was calculated for each technical replicate and the resulting mean of each sample was normalized to the empty vector control and p53 or p63 wildtype to yield the relative activity for each biological replicate.
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