FOXO-driven transcriptional activity measured by Luciferase production from different reporter gene constructs. A. Schematic diagram of the luciferase reporter constructs used in this study. One to six copies of the DBE binding cassettes were cloned upstream of the luciferase reporter gene in the pGL3-Promoter vector to create the plasmids pGL-1xDBE, pGL-2xDBE, pGL-3xDBE, pGL-4xDBE, pGL-5xDBE and pGL-6xDBE. The empty pGL3-Promoter vector and a reporter plasmid that carries three copies of a mutated DBE (mDBE) region were used as control plasmids. B. Each construct was transiently co-transfected with plasmids encoding FOXO3a and Renilla luciferase into U2OS cells, and the luciferase activities were determined as described in the "Methods" section. The data were normalized to the Renilla luciferase (phRG-TK vector) reporter construct and expressed relative to the normalized activity of the pGL3-Promoter vector. The results are given as the mean ± SEM of three independent experiments performed in triplicate. C. Basal and induced FOXO-dependent transcriptional activity conferred by three or six copies of the DBE binding cassette (grey and black bars, respectively). The plasmids pGL-3xDBE or pGL-6xDBE were transiently co-transfected into U2OS cells with plasmids encoding wild type FOXO3a or the constitutively active FOXO3a-A3 and Renilla luciferase. Where indicated cells were treated with 20 μM LY294002 or insulin for 6 hours.