High-throughput Screening for p53 Activators

A375 cells were stably transfected with the expression vector pGL4.38 [luc2P/p53 RE/Hygro] using TurboFect, as was described previously [37 (link)]. A375-p53-Luc cells were plated in white 384-well plates (Corning) at a density of 2,500 cells/25 μl/well using a Multidrop Combi dispenser (Thermo Scientific) and cultivated overnight. Library compounds were then added using pintool (V&P Scientific) coupled to a JANUS Automated Workstation (PerkinElmer) to a final concentration of 1 μM. The compound library included the Library of Pharmacologically Active Compounds (LOPAC1280, Sigma-Aldrich, Prague, Czech Republic), the Prestwick Chemical Library (Illkirch, France), and the NIH Clinical Trial Collection (NIH, Bethesda, Maryland, USA). A total of 2448 unique compounds were used in the course of HTS screening. Drugs were tested alone and in combination with a DNA damaging drug doxorubicin. The cells were cultivated for 24 h, luciferase production was determined by the One-GLO assay (Promega), and luminescence acquired by an EnVision microplate reader (PerkinElmer).