SH-EP/FOXO3 cells were treated for eight hours (a,b) or 24 hr (c,d) with 100 nM 4OHT alone or in combination with 50 µM S9 or S9OX. Cell lysates were subjected to immunoblot analyses using antibodies against NOXA and BIM (8 hr) or SESN3 and DEPP1 (24 hr). Shown are representative immunoblots (a,c) or densitometric analyses of three independent cell lysates mean values + SD. Regulations are expressed as fold over DMSO-control (100%). Significant differences between 4OHT treatment and S9+4OHT or S9OX+4OHT were analyzed by students t-test: *p<0.05, **p<0.01, ***p<0.001; two-tailed. (e) ChIP analyses were performed in SH-EP/FOXO3 cells treated with 100 nM 4OHT for three hours alone or in combination with 50 µM S9. Binding of FOXO3 to the promoter regions of BIM, NOXA, SESN3, and DEPP1 was measured by quantitative PCR. Shown is the mean value + SD of three independent experiments, each performed in duplicates. Significantly different to untreated cells: *p<0.05, **p<0.01, ***p<0.001, two-tailed. (f) Binding of FOXO3 to the promoter region of DEPP1 was assessed after transfection of a DEPP1-luciferase reporter plasmid into SH-EP/FOXO3 cells. 24 hr after transfection cells were seeded into 24 well plates. After adherence for another 24 hr, cells were treated for three hours with 100 nM 4OHT with or without increasing amounts of S9 (preincubated for 30 min). Firefly-luciferase was analyzed using the Luciferase Assay System (Promega). The increase of light emission (relative light units, RLU) was calculated as percent of untreated controls. Shown are mean values + SD of four independent experiments, each performed in triplicate. Significant differences between 4OHT treatment and S9+4OHT: *p<0.05, **p<0.01 (students t-test, two-tailed).