Recombinant murine wnt3a

Colon crypts were resuspended in ice-cold Matrigel (Corning, Cat. 356231) and allowed to solidify at 37°C, then were supplied with culture medium (750 ng/ml recombinant human Rspondin-1 (lab-made), 100 ng/ml recombinant murine Noggin (PeproTech, Cat. 250-38), 50 ng/ml recombinant murine EGF (PeproTech, Cat. 315-09), 10 μM CHIR99021 (Tocris, Cat. 4423/10), 100 ng/ml recombinant murine Wnt-3a (PeproTech, Cat. 315-20), N2 (Thermo Fisher Scientific, Cat. 15410294), B27 (Thermo Fisher Scientific, Cat. 11500446), 1X Penicillin-Streptomycin (Thermo Fisher Scientific, Cat. 15140122) in Advanced DMEM/F12 (ThermoFisherScientific, Cat. 11550446)). Organoids were cultured for 7 days, passaged via re-suspension in ice-cold PBS, then cultured for an additional 3 days before being collected in QIAzol (Qiagen, Cat. 79306) for RNA isolation.

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.

The amplified FNDC3B short or long 3′‐UTR sequences were inserted downstream of the luciferase gene in psiCHECK vector (Promega) to construct luciferase reporter plasmids. According to the manufacturer’s recommendation, the luciferase reporter plasmids of FNDC3B with short or long 3′‐UTR, plus each of 10 selective miRNA (let‐7a‐5p, miR‐17‐5p, miR‐19a‐3p, miR‐20a‐5p, miR‐34c‐5p, miR‐93‐5p, miR‐106b‐5p, miR‐125a‐5p, miR‐449a, or miR‐1224‐5p) or miRNA control mimics (RiboBio) were cotransfected into SUNE‐1 and HNE‐1 cells using Lipofectamine 3000 (Invitrogen). After 24 hours, luciferase activities were detected with the Dual Luciferase Reporter Assay System (Promega), and the firefly luciferase signal was normalized to the Renilla signal.
For Wnt reporter activity assay, the pGMTCF/LEF1‐Lu and pGMR‐TK plasmids (Genomeditech) were cotransfected into SUNE‐1 and HNE‐1 cells, together with shFNDC3B plasmid or its vector, or FNDC3B overexpressing plasmid with short or long 3′‐UTR or its vector, as well as shFNDC3B plasmid with MYH9 expressing plasmid or its vector. After 24 hours, recombinant murine Wnt‐3a (PeproTech) was added into the medium and incubated for 24 hours. Then the luciferase activities were detected, and the firefly luciferase signal was normalized to the pGMR‐TK signal.