Figure 6

REP1 negatively regulates the transcriptional activity of FOXO3 and the expression of FOXO3 target genes. (a and b) HCT116 cells were transfected with siRNAs targeting GFP or REP1. (a) FOXO3 transcriptional activity was determined using the FHRE-Luc reporter system. The cells were transfected with the FHRE-Luc reporter. A vector expressing β-galactosidase was co-transfected to ensure transfection efficiency and to normalize luciferase activity values. For analysis of the FOXO3 reporter activity in the cells, it was normalized to the control cells transfected with the empty vector. (b) mRNA expression of BAK, BIM, and p27 was analyzed by real-time quantitative RT-PCR. (c) FOXO3 transcriptional activity was determined using the FHRE-Luc reporter system. HEK293 cells were transfected with the FHRE-Luc reporter, together with the indicated plasmids. (d) HEK293 cells were transfected with empty vector (no), REP1 wild-type (WT), or REP1 mutant (Mut). mRNA expression of BAK, BIM, and p27 was analyzed by real-time quantitative RT-PCR. Fold change was calculated relative to the expression level of mRNA in control cells. All graphs represent two independent experiments performed in triplicate. Error bars represent standard deviations from the mean. *P<0.01, **P<0.001