Figure 8.

FoxO1 directly bound to the FRE site in the FoxO1 promoter. (a) A 2048-bp fragment of the FoxO1 promoter was amplified with PCR using mouse genomic DNA (bases 78–2125, 5'-UTR of FoxO1) containing a FRE (AGTAAACAAA) and cloned into the pGL3-Basic plasmid as pGL3-FoxO1 (W). pGL3-FoxO1 (M) was obtained by introducing four mutations (italics) into the FRE site. (b) FoxO1 reporter activities in mouse 3T3 cells co-transfected with FoxO1 expression vectors and FoxO1 promoter constructs for 36 h. The reporter activities were normalized to those of pRL-TK. RLA, relative luciferase activities. (c–e) The binding of FoxO1 to the FoxO1 promoter in MGCs or 3T3 fibroblasts was detected with ChIP assays following FSH treatment for 6 h. DNA was isolated from the precipitated complexes as a template for qRT-PCR (c). The qRT-PCR products were then analyzed on a 2% agarose gel (d) and quantified with densitometry using ImageJ 1.42q software (e). The amount of immunoprecipitated DNA for each ChIP reaction is shown as the percentage of input chromatin. IgG was the negative control. The data represent the means±S.E. (n=3). *P<0.05; **P<0.01, Student's t-test