Figure 1.

HuR upregulates FOXO1 expression. (A) Schematic representation of the FOXO1 mRNA 3′ UTR sequence. The HuR binding sequence ATTTA is underlined. (B) MDA-MB-231 cells were seeded in 24-well plates and transfected with 400 ng of WT or Mutant FOXO1 3′ UTRs in combination with increased doses of pcDNA-flag-HuR (100, 200 and 400 ng; n≥3). Activity of the firefly luciferase was normalized to that of the renilla luciferase. Values are expressed as means ± SEM of at least three independent experiments. ^*P<0.05. (C) MDA-MB-231 cells were seeded in 100 mm dishes. After 24 h, cells were lysed and incubated with either anti-HuR or a nonspecific IgG antibodies and Protein A Sepharose^®. Cytoplasmic extract not incubated with an antibody was saved as an ‘input’ sample. The immunoprecipitated RNAs were isolated and FOXO1 mRNA was amplified using RT-PCR. A nonspecific antibody was used as a negative control (IgG). Result presented is a representative of three different experiments. HuR, human ELAV/Hu protein; WT, wild type; M, DNA marker; FOXO1, Forkhead box protein O 1; UTR, untranslated region; RT-PCR, reverse transcription-PCR.