Runx site mutation loss of AML/ETO9a leukemic stem cells. (A) Experimental outline: WT + A/E9a and PU.1-URE-mRunx + A/E9a leukemic cells from moribund primary recipients (see Figure 6 and supplemental Figure 5) were isolated and transplanted as indicated in limiting dilutions to secondary recipients in which development of leukemia was monitored. (B) Logarithmic plot showing the percentage of negative recipients as a function of the dose of transplanted A/E9a leukemia cells. LICs were calculated as for CRUs. (C) Table showing the frequency of LICs and the total number of transplantations per cell dose. Leukemia development was monitored by survival and evaluated in spleen and bone marrow. No GFP⁺ cells were detectable in bone marrow or spleen of surviving mice after 4 months, which were then considered as nonresponders. (D) Representative picture of a leukemic (WT + A/E9a) and a nonleukemic (PU.1-URE-mRunx + A/E9a) spleen at 4 weeks after transplantation. (E-F) Flow cytometry analysis of spleens shown in (D) and corresponding bone marrows.