AML1-ETO function is impaired by mutations that disrupt DNA or CBFβ binding. (A) Diagram of AML1-ETO and location of the mutations affecting DNA (R174Q) and CBFβ binding (T161A and Y113A/T161A). (B) Lin⁻ BM cells infected with MigR1 retroviruses expressing AML1-ETO and mutated derivatives after 7 days of culture in the presence of IL-3, IL-6, SCF, and G-CSF. EGFP⁺ gated cells (not shown) were analyzed for Gr-1 and Mac-1 expression. (C) Summary of data illustrated in panel B compiled from 2 experiments, each with triplicate samples. **P ≤ .01 compared with AML1-ETO (#); ***P ≤ .001 (Dunnett test and analysis of variance [ANOVA]). (D) Serial replating analysis. Graphs represent the average number of colonies from each round of replating in the presence of IL-3, IL-6, and SCF. Week 1 represents colony numbers per 10³ cells plated, weeks 2 and 3 are from 10⁴ plated cells. Numbers are averaged from 2 experiments, each containing triplicate samples. (E) Western blot analysis probed with an antibody to the Runt domain, demonstrating expression of AML1-ETO and its mutated derivatives in MigR1-transduced NIH3T3 cells. (F) BrdU incorporation 48 hours after transduction of Lin⁻ BM cells with AML1-ETO and its mutants. EGFP⁺ cells were analyzed after a 1 hour BrdU pulse for BrdU and 7-AAD incorporation. Shown is a representative of 3 experiments. (G) Average percentages of gated BrdU⁺ (S phase) cells from scatter plots in panel F (n = 3). Error bars indicate 95% confidence intervals. **P ≤ .01 compared with AML1-ETO (Dunnett test and ANOVA).