Figure 1

MN1–Fli1 induces transformation of murine HPCs generating myeloid leukemia cells with AMKL phenotype. (a) Bar charts represent first- to third-round colony numbers as assessed by retroviral transduction and transformation assays. In brief, c-kit⁺ hematopoietic progenitor cells (HPCs) were cultured overnight in RPMI 1640 medium, 10% fetal calf serum (FCS) and 2 mM l-glutamine, supplemented with 20 ng/ml stem cell factor (SCF), 10 ng/ml IL-3 and IL-6. Spinoculation was carried out by centrifugation at 800 g (32 °C, 2 h) with 5 μg/ml polybrene (Sigma-Aldrich, Saint Louis, MO, USA). Cells were plated in M3234 methylcellulose medium (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with recombinant murine 20 ng/ml SCF, 10 ng/ml IL-3, IL-6 and granulocyte macrophage colony-stimulating factor (GM-CSF) (PeproTech EC, London, UK) and antibiotic on the following day. Colonies were scored and replated weekly. Retroviral plasmids were generated by direct cloning or PCR-based site-directed mutagenesis and verified by DNA sequencing. Error bars represent s.d. from at least three independent experiments. (b) Typical morphology of third-round colonies from primary murine bone marrow (BM) cells transduced with MN1 or MN1–Fli1. (c) May–Giemsa staining of cytospins of primary BM cells transformed with MN1 or MN1–Fli1. (d) Immunophenotype analysis of primary BM cells transformed by MN1 or MN1–Fli1. Fluorochrome-conjugated monoclonal phycoerythrin (PE) or fluorescein isothiocyanate (FITC) conjugated antibodies to murine CD41 (clone MWReg30) and CD61 (clone 2C9.G2) (Pharmingen BD Biosciences, San Diego, CA, USA) were used. Protocols and reagents used were as previously described.¹⁴ Histographs represent staining obtained with antibodies specific for murine CD41 and CD61. Error bars represent s.d. from at least three independent experiments. Two-tailed Student’s t-test was used to determine statistical significance (**P<0.05).