Evaluating CBX-mediated FOXO3 Inhibition

To determine CBX-mediated inhibition of FOXO3 binding to the DEPP- and BIM-promoter, a luciferase reporter plasmid containing the DEPP-promoter [38 (link)] or the BIM-promoter [39 (link)] was transiently transfected into SH-EP/FOXO3 cells using the JetPrime^® Reagent (Polyplus, Berkeley, USA) according to the manufacturer’s instructions. Subsequently the cells were cultured in the presence of 4OHT alone, or in combination with increasing concentrations of CBX for eight hours. Luciferase activity was measured with the Luciferase Assay System kit (Promega, Madison, USA) according to the manufacturer’s instructions. The luminescence signal was measured with the chameleon plate reader (Hidex, Turku, Finland).