H4436 methocult

Hematopoietic colony forming potential was assessed by combining cells of interest with 1.5 ml serum containing H4436 Methocult (Stem Cell Technologies). Cell suspensions were then transferred to 35-mm dishes, and cultured for 12–14 days at 37 °C. Hematopoietic colonies were scored according to cellular morphology, and CFC numbers were normalized to the number of cells plated (CFCs/10⁴) cells.

To assess colony-forming capacity of healthy CD34+ hematopoietic stem and progenitor cells (HSPC) after incubation with compound P01F08, colony forming unit assays (CFU) were performed. Therefore, 1 × 10⁵ MNC were incubated for 24 and 72 h on a 96-well plate with P01F08 in 4 different concentrations (0.3, 1, 3, 10 μM) and under previously mentioned cell culture conditions. Incubations with medium only, DMSO (0.1% v/v), and staurosporine (STS, 10 µM) were used as controls.
After incubation, viable cells were determined by the CASY cell counter. Then, 50,000 viable cells were seeded in semisolid ready-to-use methylcellulose growth medium (MethoCult H4436; Stem Cell Technologies, Vancouver, BC, Canada) in a 24-well plate and incubated for 14 days at 37 °C and 5% CO₂ under humidified conditions. Subsequently, the colonies were counted and differentiated in red precursors (colony-forming unit-erythroid, CFU-E; burst-forming unit-erythroid, BFU-E), white precursors (colony-forming unit-granulocyte, -monocyte, CFU-GM; colony-forming unit-granulocyte; CFU-G; colony-forming unit-monocyte, CFU-M) and colony-forming unit-granulocyte, -erythrocyte, -monocyte, -megakaryocyte (CFU-GEMM) by light microscopy (Axiovert 25 microscope Zeiss, Jena, Germany).

BM cells isolated from Fbxo22^+/+ and Fbxo22^−/− or Scl-Cre⁻;Fbxo22^fl/fl and Scl-Cre⁺;Fbxo22^fl/fl mice were plated into 12-well plates containing MethoCult M3434 (StemCell Technologies) for colony-forming unit-granulocyte, erythrocyte, monocyte and megakaryocyte (CFU-GEMM) or MethoCult M3534 (StemCell Technologies) for colony-forming unit-granulocyte and macrophage (CFU-GM) colony formation analysis at 2.5 × 10⁴ cells/well, MethoCult M3334 (StemCell Technologies) for colony-forming unit-erythrocyte (CFU-E) or MethoCult M3630 (StemCell Technologies) for colony-forming unit-pre-B lymphocyte (CFU-Pre-B) colony formation analysis at 2.5 × 10⁵ cells/well according to the manufacturer’s protocols. GFP⁺ or LGMP cells sorted from primary AML recipients were plated into 12-well plates containing complete MethoCult M3534 medium at indicated numbers according to the manufacturer’s instructions. For the colony-forming assay of THP-1, FBXO22-knockdown THP-1 cells or control cells were plated into 12-well plates containing complete MethoCult H4436 (StemCell Technologies) at 500 cells/well. For the colony-forming assay of AML patients’ mononuclear cells (MNCs), FBXO22-knockdown or overexpressed cells with control cells were plated into 24-well plates containing MethoCult H4436 at 40,000 cells/well. Colonies were imaged and counted 7–10 days after plating.