M3534

LSKs were plated in triplicate in methylcellulose-based medium with recombinant cytokines (without erythropoietin [EPO]) (STEMCELL Technologies, M3534) supplemented with EPO. Different types of CFUs were counted after culturing for 7–10 days.

Mouse primary MLL-AF9 AML cells were generated during a previous study [25 (link)]. Culture and transduction of MLL-AF9 AML, c-Kit⁺ BM and LSK cells were done as previously described [25 (link)]. THP-1 and MV4-11 cells were obtained from DSMZ (http://www.dsmz.de/home.html) and were cultured according to supplier’s protocol. Transduction was done with retro- or lentivirus using the RetroNectin®-bound Virus (RBV) infection method according to manufacturer’s protocol (Takara). Selection was with 2 μg/ml of puromycin for 48–72 hours prior to start of assay and then maintained. For colony forming assays, methylcellulose media M3534 and M3434 were used (for MLL-AF9 and LSK respectively, STEMCELL Technologies).

Total bone marrow cells, HSCs, YFP⁺ leukemia cells or YFP⁺Mac-1⁺c-Kit⁺ LICs were sorted by flow cytometry for the isolation of total RNA. First-strand cDNA was reverse transcribed using M-MLV reverse transcriptase (Promega Inc.). The PCR reactions were performed according to the manufacturer's protocol. The mRNA level was normalized to the level of the β-actin RNA transcripts. The primer sequences used are shown in Supplementary Table 1. For the colony-forming unit assays, the indicated numbers of cells from AML mice were plated in methylcellulose (M3534, Stem Cell Technologies) according to the manufacturer's instructions. The numbers of colonies were calculated 8–10 days after culture.