Runx1 and Cbfβ are downregulated in Mll-Af9 knock-in mice. (A) Endogenous Runx1 and Cbfβ proteins were determined by western blotting in LSK (Lin⁻ c-kit⁺ sca1⁺) populations selected by AutoMac from wild-type (WT) or Mll-Af9 knock-in C57 mice. (B) The band densities of Runx1 and Cbfβ in panel A were quantified relative to β-Actin band intensity. Each bar indicates data from 3 separate experiments. (C) Summary of relative Runx1 and Cbfβ mRNA levels in the mice LSK cells shown in panel A, which were measured by quantitative real-time polymerase chain reaction. All data were normalized to the expression of Gapdh. (D) BM cells from WT, Runx1^+/−Cbfβ^+/−, Rx1^Δ/Δ (Runx^flox/flox/Mx1-Cre⁺ with polyinosinic:polycytidylic acid injected), and Mll-Af9 knock-in mice were analyzed with M3434 methylcellulose-based medium that was formulated to support optimal growth of erythroid progenitors (burst forming unit-erythroid), granulocyte-macrophage (GM) progenitors (CFU-GM, CFU-M, CFU-G), and multipotential granulocyte, erythroid, macrophage, and megakaryocyte progenitors (CFU-GEMM). A total of 1 × 10⁴ BM cells were plated in triplicate in M3434. Colony count scoring and replating were repeated every 7 days (*P < .05; **P < .01). (E) Proportion of CFUs in serial replatings in panel D.