Meis1 transforms myeloid progenitors in synergy with Hoxa9 independently of Flt3 expression. (A) Myeloid clonogenic assay performed on Flt3^−/− or Flt3^+/+ progenitors infected with the MIE control vector (MSCV-IRES-eGFP), Hoxa9 (MSCV-hoxa9-IRES-eGFP), or Hoxa9/Meis1 (MSCV-Hoxa9-IRES-Meis1). The number of colonies are shown per 10³ (primary passage) or 10⁴ seeded cells. Results are the mean ± SEM of 3 independent experiments. The colony assay was performed as previously described.¹¹  (B) Flt3 expression in cell extracts from pooled primary colonies. Flt3^+/+ cells infected with MSCVneo, Meis1 (MSCVneo:Meis1), or Hoxa9 (MSCVneo-Hoxa9) were seeded in selective media containing G418, whereas Flt3^+/+ cells infected with Hoxa9/Meis1 (MSCV-Hoxa9-IRES-Meis1) or infected Flt3^−/− cells were grown without antibiotic. The blot was stained with FLT3 (rabbit polyclonal SC-340; Santa Cruz Biotechnology, Santa Cruz, CA) or β-tubulin (mouse monoclonal; SIGMA, Saint Louis, MO) antibodies. Bands indicated * are nonspecific. (C) Survival curves showing the fraction of Hoxa9/Meis1 Flt3^−/− (n = 6), Hoxa9/Meis1 Flt3^+/+ (n = 5), Hoxa9 Flt3^−/− (n = 6), and Hoxa9 Flt3^+/+ (n = 5) mice alive following transplantation. (D) Leukemias induced by Hoxa9 and Hoxa9/Meis1 express variable amounts of Flt3. Western-blot analysis of BM from several Hoxa9/Meis1 (A9M) and Hoxa9 (A9) leukemic mice hybridized with the antibodies directed against FLT3 or β-tubulin. BM from mice A9M#1 engrafted with Hoxa9/Meis1-transduced Flt3^−/− cells is shown as a negative control for Flt3 expression.