FIGURE 6.

MDM2 suppresses the expression of FOXO1 target gene expression and protects cells from FOXO1 induced apoptosis. A, stable expression of MDM2 suppresses the expression of FOXO target genes. Whole cell extracts of H1299 cells and MDM2 stable clones were subjected to immunoblotting with the indicated antibodies. B, MDM2 siRNA enhances the induction of TRAIL by FOXO1. H1299 cells were transfected with siRNA against GFP or MDM2. 48 h posttransfection, cellular extracts were prepared and subjected to immunoblotting with the indicated antibodies. C, MDM2 protects cells from FOXO1-induced apoptosis, as measured by manual counting. H1299 cells were transfected with 0.2 μg of pLNCE and 0.5 μg of FLAG-FOXO1 together with or without 1 μg of MDM2. Then the cells were fixed and stained with DAPI. Representative micrographs were captured by a CCD camera attached to a fluorescence microscope (upper panels). The apoptotic index of GFP-positive cells was determined by scoring 300 GFP-positive cells for chromatin condensation and nuclear fragmentation. Triplicate samples were analyzed per data point, and the graph represents three independent experiments. D, MDM2 protects cells from FOXO1-induced apoptosis, as measured by flow cytometry. H1299 cells were transfected with GFP-spectrin and FLAG-FOXO1 with or without MDM2. Transfected (GFP-positive) cells were separated from nontransfected (GFP-negative) cells by FACS-based sorting. The apoptotic index of GFP-positive cells was determined with the Annexin-V kit. The flow cytometry profile of a representative experiment was shown. Data from two independent analyses are shown as bar graphs. MnSOD, manganese superoxide dismutase.