Figure 7.

PP2A is required for efficient nuclear translocation and transcriptional activation of FOXO3a following PI3K/AKT inhibition. (A) GFP-FOXO3a expression plasmid was cotransfected into HeLa cells with either an EV or ST expression plasmid. Cells grown in serum were either fixed (−) or treated with 20 μM each of LY and AKT-I for 30 min in the absence of serum (+) before fixation. GFP-FOXO3a and 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei were visualized by fluorescence microscopy. (B) HeLa cells were transfected with either an EV or ST expression plasmid. Cells grown in serum were either fractionated (−) or treated with 20 μM each of LY and AKT-I for 30 min in the absence of serum (+) before fractionation. Cytoplasmic (CE) and nuclear (NE) extracts were immunoblotted (IB) with the anti-IκBα and -Sp1 antibodies to evaluate extract purity. (C) FOXO3a was immunoprecipitated (IP) from whole-cell lysates derived from HeLa cells transfected with wild-type FLAG-FOXO3a (WT), FLAG-FOXO3a-TM (TM), or FLAG-FOXO3a-TM and ST (TM/ST) expression plasmids. Immunocomplexes were IB with the indicated antibodies. (D) A GFP-FOXO3a- TM expression plasmid was cotransfected into HeLa cells with either an EV or ST expression plasmid. GFP-FOXO3a-TM and DAPI-stained nuclei were visualized by fluorescence microscopy. (E) Analysis of FLAG-FOXO3a-TM subcellular localization by fractionation. (F) BaF3/pMIG (EV) and BaF3/pMIG/ST (ST) cells were grown in the presence of interleukin (IL)-3 and either lysed (−) or treated with 20 μM each of LY and AKT-I for 7h in the absence of IL-3 (+) before lysis. Quantitative real-time RT-PCR was used to calculate the mean fold induction in bim (left) and trail (right) transcripts after LY/AKT-I treatment relative to untreated BaF3/pMIG cells, which is shown by gray columns. Results shown are a representative of three independent experiments performed in duplicate (black and white columns) each time. Error bars represent the SD from a representative experiment. (G) HeLa cells with a chromosomally integrated luciferase reporter plasmid containing six-tandem FRE from the human TRAIL promoter were transfected with the indicated expression plasmids (6X-FRE/TRAIL-Luc). The increase in firefly luciferase activity is shown as the mean fold induction over the activity in cells transfected with EV alone. In each experiment, triplicate dishes for each transfection group were analyzed. The result presented in this figure is a representative of two independent experiments. Each column represents the mean of the triplicate values, and the error bars represent the SD of the mean for this particular experiment (top). Cell extracts from the presented reporter experiment were analyzed by immunoblotting with the indicated antibodies (bottom). (H) The HeLa reporter cell line was transfected with an empty vector (EV), wild-type FLAG-FOXO3a (WT), or FLAG-FOXO3a-TM (TM) expression plasmids together with either nontargeting siRNAs (NT) or siRNAs targeting the PP2A catalytic subunit (Cα) as indicated. Results were analyzed as described in G.