In vivo activities of AML1-ETO are relatively unaffected by mutations that impair HEB binding. (A) Schematic diagram of AML1-ETO and the location of mutations (asterisks). (B) Effect of eTAFH mutations on AML1-ETO's repression of granulocyte differentiation after 7 days of culture in the presence of IL-3, IL-6, SCF, and G-CSF. Cells within the forward and side scatter gates were further gated for GFP expression, and GFP-positive cells were examined for Mac-1 and Gr-1 expression. Data represent triplicate samples from 2 independent experiments. Error bars represent 95% confidence intervals. Significant differences from AML1-ETO (#) are indicated with asterisks (ANOVA and Dunnett multiple comparison test, **P < .01). (C) Representative flow of Lin⁻ bone marrow cells infected with MigR1 retroviruses expressing AML1-ETO, the m7 oligomerization mutant, and the m7+eTAFH mutants. (D) Average percentages of Gr-1⁺Mac-1⁺ cells. Significant differences relative to the m7 mutant are indicated with asterisks (ANOVA and Dunnett multiple comparison test, **P < .01, *P < .05). (E) Cos7 cells were cotransfected with AML1/ETO and its mutated derivatives and FLAG-tagged HEB. (Top) Cell lysates immunoprecipitated (IP) with anti-FLAG and blotted with antibody to the Runt domain (RD) in AML1/ETO. (Middle) Input lysate (1%) was blotted with anti-RD to detect AML1/ETO proteins. (Bottom) Membranes from the top panel were reprobed with anti-FLAG antibodies. The percentages of immunoprecipitated AML1-ETO proteins relative to input were 3.7% (AML1/ETO), 2.3% (AML1-ETO F332A), 1.1% (AML1-ETO m7), and 0.7% (AML1-ETO m7+F332A). (F) Representative flow of BrdU incorporation 48 hours after transduction of Lin⁻ bone marrow cells with MigR1 expressing GFP, AML1-ETO, or the AML1-ETO eTAFH mutants. (G) Percentage of GFP⁺ cells that had incorporated BrdU after an 1-hour BrdU pulse. Data are from 2 experiments each with triplicate samples (error bars = 95% confidence intervals; significant differences from AML1-ETO indicated with an asterisk, ANOVA and Dunnett multiple comparison test, **P < .01). (H) Serial replating of bone marrow cells. Graphs represent the average number of colonies from each round of replating in the presence of IL-3, IL-6, and SCF. Day 7 represents colony numbers per 10³ cells plated and days 14, 21, and 28 from 10⁴ plated cells. Numbers are averaged from 3 experiments, each containing triplicate samples. The numbers of colonies derived from F277A- and F332A-transduced cells were significantly lower than those from AML1-ETO–transduced cells at day 14 and day 28 (P < .01). Error bars represent 95% confidence intervals.