Figure 2

DEPP is a direct transcriptional target of FOXO3. a) Quantitative RT-PCR of DEPP expression in SH-EP/FOXO3 cells, treated with 75 nM 4OHT for 2 hours to activate FOXO3(A3)ERtm or a combination of 4OHT and cycloheximide (CHX; 10 μg/ml), which was used to block protein synthesis. Shown are mean values ± s.e.m. of three independent experiments, each performed in triplicates. b) The DEPP gene promoter (-1116 bp relative to the transcription start) contains three putative binding sites for FOXO3, which are highlighted in the schematic representation. A DEPP wild type promoter reporter plasmid and DEPP promoter reporter plasmids containing mutations of the three FOXO3-binding sites (B1, B2 and B3; indicated as bold in sequence) were transfected into SH-EP/FOXO3 cells. The cells were treated with 100 nM 4OHT for 4 hours to activate FOXO3(A3)ERtm and a luciferase-assay was performed. Direct binding of FOXO3 to the DEPP promoter leads to increased luciferase activity. The increase of the luciferase signal was calculated as fold over untreated controls. Shown are mean values ± s.e.m. of three independent experiments, each performed in duplicates; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05; **P < 0.025 compared to corresponding controls. c) ChIP analysis on the interaction between FOXO3 and the FOXO3 binding sites B1 + 2 and B3 of the DEPP promoter in SH-EP/FOXO3 cells treated with 100 nM 4OHT for 3 hours. Quantification was performed by quantitative RT-PCR with specific primers for B1 + 2 and B3. Shown are mean values of two independent experiments, each performed in duplicates.