Nitrotetrazolium blue

Animals were deeply anesthetized with isoflurane and decapitated before the gastrocnemius muscles were dissected. Muscles were embedded in OCT compound (Fisher Scientific) and placed on dry ice. Sections were cut at 14μm on a cryostat and placed directly onto coverslips. For hematoxylin & eosin (H&E) staining, muscle sections were stained with hematoxylin for 5 minutes, followed by three 10-minute washes in deionized H₂O. Muscles were then placed in eosin G for 1 minute, then rinsed in 70% ethanol. The tissue was then dehydrated in a series of ethanol washes (70%, 95%, 100%), rinsed in xylene, then mounted on a glass slide. NADH staining was performed by incubating muscle sections in 0.2M Tris buffer containing Nitrotetrazolium Blue (Sigma) and β-nicotinamide adenine dinucleotide (NADH, Sigma) for 30 minutes at 37°C. Following three washes in deionized H₂O, coverslips were mounted onto a glass slide and imaged using an Olympus microscope (BX-51).

Cell lines were cultured (10,000 cells/sample) in 0.35% Noble agar on a 0.5% Noble agar base layer and overlaid with cRPMI containing kinase inhibitor or vehicle. The overlying medium was replaced 2–3 times per week, and vehicle treatment was assessed in duplicate. After 14 days or 21 days (Kasumi-1 cells only), colonies were stained with 1 mg/ml nitrotetrazolium blue (Sigma-Aldrich) for 4 hours and counted using a GelCount colony counter (Oxford Optronix). Mononuclear cells were isolated from human cord blood and samples from AML patients using Ficoll-Paque PLUS (GE Healthcare Life Sciences). Patient samples were cultured in triplicate at a density of 1 × 10⁶ cells/ml in MethoCult H4434 Classic Methylcellulose-Based Medium with Recombinant Cytokines for Human Cells (STEMCELL Technologies) containing MRX-2843 or vehicle. Colonies were counted after 10 days using the GelCount colony counter (Oxford Optronix). Cord blood cells were incubated for 1 hour in serum-free Iscove’s modified Dulbecco’s medium (IMDM) (HyClone) supplemented with BIT 9500 Serum Substitute (STEMCELL Technologies), low-density lipoproteins (EMD Millipore), and 2-ME (Sigma-Aldrich), and then cultured in triplicate at a density of 2 × 10⁶ cells/ml in Methocult H4434 methylcellulose containing MRX-2843 or vehicle. Colonies were manually counted in a blinded manner after 14 days.