FIGURE 1.
Conditional FOXO3 activation induces apoptosis in primary ECs irrespective of direct FRE-binding. HUVECs were retrovirally infected with either empty vector, FOXO3.A3.ER or FOXO3.A3.ER.H212R, selected by puromycin, and reseeded. A, luciferase assay demonstrating the incapability of the H212R mutant to transactivate a forkhead-responsive 6×DBE-luc reporter upon 16-h treatment with 4OHT. Data represent -fold average luciferase activation ± S.D. of a 6× DBE-luc reporter transfected into the differently infected cells. Luciferase values were each normalized to activity of a co-transfected Renilla luciferase reporter and related to unstimulated empty vector-infected cells. B, HA tag-specific chromatin immunoprecipitation after 12 h of 4OHT treatment showing binding of HA-FOXO3.A3.ER but not HA-FOXO3.A3.ER.H212R to an established FOXO-responsive region (6) in the IGFBP1 promoter. Data are represented as -fold enrichment of binding related to 4OHT-treated vector controls. C, qRT-PCR demonstrating expression of IGFBP1, normalized to GAPDH expression after 12 h of 4OHT treatment. Data display average -fold regulation ± S.D. related to unstimulated vector. D, Western blot, showing expression of p27Kip1 and cleavage of caspase-3 32 h after 4OHT treatment. E, cell cycle profiles, displaying the percentage of subdiploid DNA content assessed by flow cytometric analysis of PI-stained cells 48 h after 4OHT treatment. F, colony formation assay after 6 days (6d) of 4OHT treatment. All data are derived from at least three independent experiments. Statistical significance was calculated by Student's t test.