Expression of p19^ARF is regulated by AML1 in MLL-ENL leukemia. (A) Expression analyses of p19^ARF in MLL-ENL leukemic cells. Aml1 intact or Aml1-excised MLL-ENL leukemic cells harvested from each spleen were transduced with GFP or AML1-GFP. Forty-eight hours later, expression levels of p19^ARF were measured by quantitative real-time PCR. (B) Aml1 intact MLL-ENL leukemic cells were transduced with p19^ARF shRNA or control shRNA and transplanted into secondary recipient mice. Survival curves of 12 mice from each group are shown. Comparison of survival curve was performed using log-rank test. (C) Expression levels of p19^ARF in leukemic cells from secondary recipient mice were measured by quantitative real-time PCR. (D) Two AML1 binding sites located in the promoter of p19^ARF are shown as indicated. (E) Chromatin immunoprecipitation analyses of AML1 for p19 ^ARF promoter region in MLL-ENL leukemic cells. Fold enrichment normalized to the control locus Gapdh was shown. (F) COS7 cells were cotransfected with expression vector for AML1 and wild-type or PEBP2-site mutated p19^ARF promoter vector. The relative luciferase activity was calculated as the ratio of luciferase activity with AML1 expression to that without AML1 expression. All luciferase reporter assays were performed in duplicate in 2 independent experiments. Values and error bars represent the mean and the SD, respectively. *P < .05. We performed 3 independent experiments and confirmed that similar results were reproduced, except for panel B. Statistical significance was evaluated by unpaired t test.