Figure 3.

FO1-6nls augments FOXO1's transcriptional activity and antagonizes CDK1-induced nuclear exclusion of FOXO1. (A) The antagonistic effect of FO1-6nls on CDK2-mediated inhibition of FOXO1's transcriptional activity. LNCaP cells were transfected with plasmids as indicated. At 36 hours after transfection, luciferase activities were measured and analyzed as described in the Materials and Methods section. Error bars, SD of three independent experiments. (B) Effect of FO1-6nls on the transcriptional activity (left) and cellular localization (right) of FOXO proteins in PTEN-null PC-3 cells. Left, PC-3 cells were transfected with FO1-6nls and/or pretreated with the PI3K inhibitor LY294002, and luciferase activities were measured as described in A. Right, PC-3 cells were transfected with plasmids as indicated and plated in serum-containing media. At 24 hours after transfection, cells were treated with LY294002 (20 µM) or vehicle for 12 hours. Cells were then subjected to immunofluorescent chemistry with antibodies indicated. (C) Effect of FO1-6nls on the transcriptional activity of endogenous FOXO1 proteins in PTEN-positive DU145 cells. Cells were transfected with the reporter gene 3xIRS-Luc and different amount of FO1-6nls. At 36 hours after transfection, luciferase activities were measured and analyzed as described in A. Error bars, SD of three independent experiments. (D) BPH-1 cells were transfected with plasmids as indicated and subjected to luciferase activity measurement as in A. (E) DU145 cells transfected with plasmids as indicated and plated in serum-free media. At 48 hours after transfection, cells were subjected to immunofluorescent chemistry with antibodies indicated.