Mil 3

Primary mouse bone marrow cells were transduced as previously described20 (link). Briefly, bone marrow cells were harvested from mice treated 4 days previously with 150 mg/kg body weight 5-fluorouracil (5-FU; Sigma, St. Louis, MO) and cultured for 24 h in Dulbecco’s modified eagle’s medium supplemented with 10% FBS, 1% penicillin/streptomycin (Gibco Technologies, Carlsbad, CA), 50 ng/mL recombinant mouse stem cell factor, 10 ng/mL mouse interleukin 6 (mIL-6), and 10 ng/mL mouse interleukin 3 (mIL-3; R&D Systems, Minneapolis, MN). The cells were then harvested and slowly spread on the top surface of the membrane of a Transwell insert (Corning Incorporated Life Sciences, Acton, MA) at a density of 2 × 10⁶ cells per well. The viral supernatants were added to the Transwell inserts along with 4 μg/ml of polybrene and were cultured with cells for a further 48 h. The viral supernatant was changed 3 times during this period of time. Retrovirally-transduced, i.e. GFP positive, bone marrow cells were flow cytometrically sorted using a FACSAriaII (Becton-Dickinson) and used for CAFC assay and transplantation assays.

To measure growth and survival of BaF3 cell populations expressing EGFR WT or EGFR^C329R, cells were washed 3 times with PBS and incubated with growth factor (mIL-3 or rhEGF; R&D Systems, Minneapolis, MN, USA) at the indicated doses. Cell growth was assessed using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) (Promega, Madison, WI, USA) following the manufacturer’s instructions. Caspase activity was measured with Caspase-Glo 3/7 Assay (Promega) as per the manufacturer’s instructions. Viability was measured by Annexin V (BD Biosciences) and 7AAD (BD Biosciences) staining, following the manufacturer’s protocol.

ES cell-derived HPCs were derived according to the protocol described previously by Chan et al. [3 ]. Briefly, HOXB4-transduced mouse ES cells (HM1 cell line) originally derived from the 129SvJ mouse strain were grown on feeder-seeded gelatinized flasks in ES cell culture medium with 15% fetal calf serum, 1% penicillin/streptomycin cocktail (GIBCO), 0.1 mM l-glutamine, and 1000 U/ml leukemia inhibitory factor for the maintenance of pluripotency. The ES cells were then subjected to embryoid body (EB) formation. The EBs were trypsinized and dissociated into a single cell dispersion before being replated onto ultralow-attachment Petri dishes in defined medium containing StemPro34 base media plus nutrient supplement (Life Technologies/BRL) and various hematopoietic cytokines: mIL-3 (2 ng/ml), mouse stem cell factor (100 ng/ml; R&D Systems), mIL-6 (5 ng/ml), IGF-1 (40 ng/ml; Promega), Flt3-L (10 ng/ml), and dexamethasone (1 μM; Sigma-Aldrich). The cell cultures were kept in a hypoxic incubator containing 9% CO₂.

CFU-GEMM, CFU-GM and BFU-E colony formation was assessed in vitro using washed suspensions of 5 X10⁴ marrow cells or 3 X 10⁵ spleen cells suspended in IMDM and plated in 1% methylcellulose with 30% FBS (Methocult medium, catalogue #3534, STEMCELL Technologies, Vancouver, BC Canada) containing 10 ng/μL of mIL3 (catalogue #02733, STEMCELL), 10 ng/μL of rhIL6 (#206-IL-010, R&D SYSTEMS, Minneapolis, MNUSA), 50ng/μL of THPO (#288-TP-005, R&D) and 3 U/μL of rhEPO (Johnson and Johnson, New Brunswick, NJ USA). All colony-forming assays were performed between weeks 15–18. Colony-forming assays were performed in triplicate with at least 3 replicate experiments and colony formation was assessed at 7 days. CFU-Mk colony formation was assessed using 5 X10⁴ washed marrow cells or 3 X 10⁵ spleen cells suspended in IMDM and plated in methylcellulose (Megacult-C medium, # 04850, STEMCELL) containing 10 ng/μL of mIL3, 10 ng/μL of rhIL6 and 50ng/μL of THPO, R&D). Colony number was assessed at 7 days after staining with acetylcholinesterase activity as described in the Megacult-C protocol for murine cells.

HEK293T (RRID:CVCL_0063) and NIH 3T3 (ATCC Cat# CRL‐1658, RRID:CVCL_0594) were from ATCC and cultured at 37 °C with 5% CO₂ in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% vol/vol fetal bovine serum (FBS) and penicillin (100 U mL⁻¹)/streptomycin (0.1 mg mL⁻¹). MOLM‐13 (RRID:CVCL_2119) was from DSMZ and cultured at 37 °C with 5% CO₂ in RPMI 1640 medium supplemented with 10% FBS and penicillin (100 U mL⁻¹)/streptomycin (0.1 mg mL⁻¹). Mouse HSPCs and AML cells were cultured at 37 °C with 7.5% CO₂ in BCM medium [45% IMDM + 45% DMEM + 10% FBS + 50 × 10⁻⁶mβ‐mercaptoethanol + penicillin (100 U mL⁻¹)/streptomycin (0.1 mg mL⁻¹)] supplemented with 10% stem cell medium (SCM) including mIL‐3 (10 ng mL⁻¹, Cat# 403‐ML; R&D Systems), mIL‐6 (10 ng mL⁻¹, Cat# 406‐ML; R&D Systems), and mSCF (50 ng mL⁻¹, Cat# 455‐MC; R&D Systems).