Analyzing AML/MDS Cell Lines

Bone marrow, peripheral blood or leukapheresis samples from patients diagnosed with AML/myelodysplastic syndromes (MDS) (for clinical information see Online Supplementary Table S1) were collected in accordance with the Declaration of Helsinki and with approval of University Hospitals Bristol NHS Trust and London Brent Research Ethics Committee. Mononuclear cells were separated using Ficoll-Hypaque (Sigmα-Aldrich, Poole, UK) and samples with ≥80% viability included in the study. K562, HEL, ML-1, U937, THP1 and PLB-985 cell lines (ECACC, Salisbury, UK) were cultured as previously described.^{11 (link)} For proliferation assays, cell lines were seeded in triplicate at 1×10⁵/mL into 24-well plates within medium containing 10, 5, 1 or 0.5% fetal bovine serum (Labtech, East Sussex, UK) and cellular density counted using a hemocytometer at 24, 48 and 72 hours (h). For Wnt signaling activation, cell lines were treated with 5 μM of the GSK-3β inhibitor CHIR99021 (Sigmα-Aldrich) or 1 μg/mL recombinant murine Wnt3a (Peprotech, London, UK) for 16 h (unless otherwise stated) at 37°C.