Myeloid Progenitor Transformation Assay

The myeloid progenitor transformation assay was carried out as previously described^{52 (link)}. Bone marrow cells were harvested from the femurs and tibiae of 5-week-old female C57BL/6 mice (purchased from CLEA Japan, Inc). c-Kit-positive cells were enriched using magnetic beads conjugated with an anti-c-Kit antibody (Miltenyi Biotec, 1:50 dilution), transduced with a recombinant retrovirus by spinoculation, and then plated in a methylcellulose medium (Iscove’s Modified Dulbecco’s Medium, 20% FBS, 1.6% methylcellulose, and 100 µM β-mercaptoethanol) containing murine stem cell factors, interleukin-3, and granulocyte-macrophage colony-stimulating factor (10 ng ml⁻¹ of each). G418 (1 mg ml⁻¹) was added to the first round of culture to select for transduced cells. Hoxa9 expression was quantified by RT-qPCR after the first round of culture. Colony-forming units (CFUs) were quantified per 10⁴ plated cells after 4–6 days in culture. This protocol was approved by the National Cancer Center Institutional Animal Care and Use Committee of the National Cancer Center, Tsuruoka, Japan.