Prl tk renilla reporter

HCT116 cells, p53-proficient or p53-deficient, at 60% confluence in 6-well plates were co-transfected with 500 ng pGL3-Basic luciferase reporter constructs containing a sequence derived from the identified p53-binding site upstream of the RECQ1 gene, (PGL3_RECQ1), wt p53 binding sites (PGL3_p53RE; a gift from Bert Vogelstein (Addgene plasmid # 16442)[41 (link)], or empty vector and 50 ng pRL-TK Renilla reporter (Promega) by Lipofectamine 2000 as described by the manufacture's protocol. Cells were lysed 36 h post-transfection and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) according to protocol. Firefly luciferase activities were normalized with Renilla luciferase activities to obtain the relative luciferase activity. Luciferase activity for pGL3_RECQ1 was comparable to that of pGL3_p53RE. Results are presented as normalized relative luciferase activity compared to p53-proficient HCT116 cells.

Cells were seeded as 2 × 10⁵ cells/well in a six-well plate and co-transfected with pMyc-Luc reporter (Signosis, Inc. Santa Clara CA, USA) and pRL-TK Renilla reporter (Promega Corporation, Madison, WI, USA) plasmids at a ratio of 50:1 with TurboFect transfection reagent (Thermo Fisher Scientific Inc.) for 24 h. After treatment of 17-DMAG for a further 48 h, cells were lysed with 200 µL passive lysis buffer (Promega) and determined the luciferase activity with Beetle-Juice (for firefly luciferase (FLuc)) and Gaussia-Juice (for Renilla luciferase (RLuc)) substrates (PJK GmbH, Kleinblittersdorf, Germany), and luminescence was counted on a luminescence reader (Promega). The c-Myc transcriptional activity was determined according to the FLuc count after normalization to RLuc, which represented the transfection efficiency of each sample.