Fig. 6.

FoxO3a regulates the cyclin A promoter. (A) Cyclin A1 promoter region contains a putative FoxO3a binding site. The response element was mutated as described in Materials and Methods for a mutant cyclin A1 promoter lacking a functional FoxO3a consensus sequence. (B) Comparison of luciferase activity of the wild-type cyclin A1 promoter with the mutant cyclin A1 promoter in KTC3 shFoxO3a and FRO wt FoxO3a cells. Cells were transiently transfected as described in Materials and Methods. The results given as average relative luminescent units (firefly activity/renilla luciferase vector activity) ± s.d. *Comparison between control and FoxO3a shRNA or wt FoxO3a within a promoter, ⁺comparison of activities across wild-type and mutant cyclin A1 promoters. (C) FRO cells were transfected with vector control or FLAG-tagged FoxO3a. FoxO3a/DNA complexes were immunoprecipitated (anti-FLAG) after crosslinking. An immunoprecipitation with mouse IgG served as a negative control (control). Precipitates were analyzed by PCR for the FoxO3a-bound cyclin A1 promoter. A PCR for the cyclin A1 promoter using the input DNA served as an additional control.