Evi1 represses PTEN protein level and activates downstream AKT/mTOR signaling. (A-B) Analysis of indicated protein levels in Evi1 or mock-transduced BM cells. BM cells (1-2 × 10⁶) were used in these assays. Experiments were repeated for > 2 times, and the representative figures are presented. (C) Comparison of PTEN mRNA expression between various oncogene-transduced BM cells. Mock, AML1/ETO, E2A/HLF, PML/RARα, or Evi1-transduced BM cells were prepared and were analyzed after 1 week of G418 selection. Error bars indicate SD (n = 4; *P < .01). Results were represented as the averages of 4 independent experiments performed in duplicate. (D) Comparison of PTEN/AKT/mTOR signaling of the indicated cells analyzed by Western blotting. BM cells (1-2 × 10⁶) were used in these assays, and 4 independent experiments were performed and representative figures are shown (n = 2 for each). (E) Rapamycin was added to each oncogene- or mock-transduced BM cells with the indicated concentrations in semisolid medium. The average colony counts were converted to percentages, defining the colony number without rapamycin as the cell viability of 100%. P values were calculated by comparing percentages of viable cells at 0.4nM rapamycin. Error bars indicate SD (n = 8 from 4 independent experiments; *P < .00001). (F) Cell cycle analysis of Evi1- or mock-transduced BM cells with/without addition of rapamycin. Representative fluorescence-activated cell sorting data (left), and average percentage of cells in G₂/M phase (right) were shown. Error bars indicate SD (n = 3; *P < .01). (G) A BM smear of Evi1-induced AML, stained with Wright-Giemsa, showed an increase of myeloblasts. Slides were examined by Olympus BH-2 microscope with 40×/0.75 NA oil objective. Picture was taken with Olympus DP20-E camera and analyzed with Adobe Photoshop 7.0. (H) Representative flow cytometric profiles of the BM cells isolated from a recipient of Evi1- or mock-transduced BM cells. The surface marker profiles of Evi1-induced leukemic cells were almost the same. These cells expressed c-kit and Mac-1. Lymphoid markers such as CD3 and B220 were negative except for some deviations in the intensity of B220. In contrast, mock-transduced cells were hardly detected, which suggested that the transplanted cells did not engraft. The numbers in the figure show the percentage of cells gated in each quadrant. (I) Survival of Evi1-induced leukemic mice (top; n = 20 in total, 3 clones were transplanted), or that of TEL/PDGFβR-AML1/ETO-induced leukemic mice and AML1_S291fsX300-induced leukemic mice (bottom; n = 20 in total for each leukemia, 2 clones were transplanted for each) treated with vehicle or rapamycin. DMSO indicates dimethyl sulfoxide; ERK1/2, extracellular signal-regulated kinase 1/2; STAT3, signal transducer and activator of transcription 3; STAT5, signal transducer and activator of transcription 5.