Figure 7.

FOXO1 suppression of basal Fshb transcription may involve PITX1. (A) The 4xHDBE-luc reporter was transiently transfected into CV-1 cells along with pcDNA3 empty vector (EV), FOXO1, and PITX1, as indicated. The results represent the mean ± SEM of three experiments performed in triplicate and are presented as fold PITX1 induction relative to pcDNA3 empty vector. *, transcription is significantly repressed by FOXO1 compared to EV using Student's t test. (B) Diagram illustrating the mutations in the PITX1 binding element (PITXBE) in the murine Fshb promoter. (C–D) The −1000 Fshb-luc reporter and the Fshb PITX1 mutant (mut) were transiently transfected into CV-1 (C) or LβT2 (D) cells along with EV, FOXO1, and PITX1, as indicated. The results represent the mean ± SEM of three experiments performed in triplicate and are presented as fold PITX1 induction relative to pcDNA empty vector (C) or luc/β-gal (D). *, Fshb-luc transcription is significantly repressed by FOXO1 compared to EV using Student's t test; #, PITX1 mut was significantly repressed compared to the wild-type Fshb promoter. (E) GST interaction assays were performed using bacterially expressed GST-fusion proteins (indicated above each lane) and ³⁵S-labeled in vitro transcribed and translated FOXO1, FOXO1-ΔDBD, and GFP (indicated on the left of the panels). GFP was used as a negative control. The GST-fusion proteins included GST alone and GST-PITX1. One-quarter of the protein used in the interaction assay was loaded in the lane marked input. The experiment was repeated several times with the same results and a representative experiment is shown.