Dot1l deletion impairs the transforming capacity of MLL-AF6–transformed bone marrow cells. (A) Differential colony counts from Dot1l-excised, MLL-AF6–transformed cells 7 days after Cre transduction compared with vector (Mi-Tomato)-expressing controls (n = 3 independent experiments). Light gray bars represent dense and compact blast-like colonies, whereas dark gray bars represent small and diffuse colonies showing marked features of differentiation. Colony-forming units (CFU)/1000 cells averaged from 3 independent experiments are plotted with standard error of the mean (SEM) values. *P value = 0.002. (B) Morphological changes in colony and cell types upon Dot1l deletion in bone marrow cells immortalized by MLL-AF6 (colonies, ×10; cell morphology, ×40). (C) Enrichment of H3K79me2 normalized to input DNA on the chromatin locus of Hoxa/Meis1 gene promoters as assessed by quantitative reverse-transcription (qRT)-PCR is shown 6-9 days after Cre transduction compared with Mi-Tomato–transduced cells (n = 2 independent experiments). (D). RT and qRT-PCR 5 days after transduction with Cre-Mi-Tomato or Mi-Tomato. Expression levels normalized to Gapdh and expressed relative to Mi-Tomato–transduced cells (set to 100%) are shown. Error bars represent the SEM (n = 2 independent experiments). (E) Survival curves for mice injected with 2 × 10⁵ wild-type or Dot1l-excised MLL-AF6 primary leukemia cells (left). Hematoxylin and eosin staining showing blast infiltration in the kidney of a representative leukemic mouse injected with Dot1l ^+/+ MLL-AF6 leukemias is presented (right). Staining from a disease-free mouse injected with Dot1l ^−/− cells and sacrificed at an identical time point after injection is shown for comparison.