Involvement of Foxo transcription factors in angiogenesis and postnatal neovascularization
J. Clin. Invest. Michael Potente, et al. 115:2382 doi:10.1172/JCI23126 [
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Figure 5Foxo1 and Foxo3a are transcriptional repressors of eNOS. (
A) eNOS expression in HUVECs that were transfected with constitutively active constructs of Flag–Foxo1 A3 and HA–Foxo3a A3. Cell lysates were prepared at the indicated time points, and expression of eNOS, Flag, HA, and tubulin was determined by Western blotting using the respective antibodies. (
B) eNOS expression in HUVECs that were transfected with Foxo1- or Foxo3a-specific siRNA and lysed at the indicated time points. eNOS, Foxo1, and Foxo3a expression was determined by immunoblotting. SCR, scrambled siRNA; FX1, Foxo1 siRNA; FX3a, Foxo3a siRNA. (
C) eNOS expression in the aorta of
Foxo3a+/+ and
Foxo3a–/– mice. After the aortas from the indicated groups of mice were removed, cell lysates were prepared, and expression of eNOS was determined by immunoblotting. Equal protein loading was confirmed with a tubulin antibody. (
D) HUVECs were transfected with Foxo1 A3 (Flag-Foxo1 A3), Foxo3a A3 (HA–Foxo3a A3), Foxo4 A3 (HA–Foxo4 A3), or vector control. After 24 hours, chromatin-bound DNA was immunoprecipitated with an antibody against the Flag or HA epitope. Immunoprecipitated DNA was analyzed by PCR using a primer combination that encompassed the forkhead responsive element (FHRE). The pGL3-eNOS-3500 plasmid was used as a positive control for the PCR. eNOS primers for the eNOS coding sequence were used as a negative control to exclude nonspecific precipitated DNA. CDS, coding sequence. (
E) HUVECs were cotransfected with pGL3-eNOS-3500 and either Foxo1 A3 or the empty vector pcDNA3 (pcDNA). Luciferase activity was measured 24 hours later. Values are expressed as the level of luciferase activity of Foxo1 A3 relative to that of pcDNA, which was set as 100%. Data are presented as mean ± SEM;
n = 6. *
P < 0.001.
