AES expression is required for AML1/ETO-induced proliferation. (A) Two different shRNAs against AES delivered by lentiviral vectors were shown to down-regulate AES expression in Kasumi-1 cells, as demonstrated in Western blot analyses stained with anti-AES antibody. Anti-actin serves as loading control. (B) Limited dilution analyses of cloning capacity of the stably transduced and sorted Kasumi-1 cells showed a significantly reduced frequency of self-renewal capacity. Relative cloning efficiency was calculated as the frequency of growing colonies in correlation to seeded cell number. The log fraction of negative wells on input cells per plate is indicated (Poisson distribution). (C) Scheme of experimental setup to analyze self-renewal capacity in primary murine BM. The transforming capacity of AML1/ETO in CD11b⁺ BM cells was used to select for cells with self-renewal capacity after several rounds of replating. Immortalized cells were subsequently infected with LKO.1-puro vectors expressing shRNA against AES, selected and replated in colony assays. (D) The ability to form colonies on replating is shown after AES knockdown compared with nontransduced or scrambled transduced (control) cells. Data are mean plus or minus SD of 2 independent experiments each performed in triplicate (total 6 dishes). Differences between AES-shRNA and scrambled control transduced colony numbers were statistically significant (P < .001).