Methocult m3534

CFU assays were performed as described previously. Murine bone marrow cells (5 × 10⁴) were plated in methylcellulose-based medium (Methocult M3534; Stem Cell Technologies, USA) with AnxA1 (100 nM) and signalling inhibitors of PLC (U73122; 5 µM/) or PKC (GF109203; 10 nM)^{53 (link)}, and total number of colonies counted after 3 days under an optical microscope at ×40 magnification (Carl-Zeiss, Germany). An additional set of experiments was also performed: here bone marrow cells were collected, treated with the inhibitors of PLC or PKC for 1 h, and subsequently stimulated with 100 nM AnxA1 for 30 min. Expression of p-ERK and p-Elk-1 was quantified by flow cytometry as above.

Colony-forming cells (CFCs) were assayed in methylcellulose (Methocult M3534; StemCell Technologies Inc.) as described previously (Heuser et al., 2007 (link)). 100 cells were mixed with 1 ml methylcellulose and inhibitor or DMSO as control. Cells were incubated at 37°C under 5% (v/v) CO₂. Colonies were evaluated microscopically 7 days after plating by using standard criteria. For replating, cells were eluted from the methylcellulose, washed, counted and replated as described above.

After sorting CD45.2⁺ murine NK and CD45.1⁺ HSC cells (LSK SLAM⁺), co-culture of NK : HSC cells in a 100:1 ratio was carried out. Cells were plated in StemSpan SFEM Medium^® supplemented with IL-2 (1,000 U/ml), SCF (100 ng/ml), IL-3 (10 ng/ml), IL-6 (10 ng/ml), FLT3 (100 ng/ml), and TPO (25 ng/ml), as well as penicillin and streptomycin (1%) for 24 h. This ratio was proposed to simulate the average proportion of NK and HSC observed in the healthy bone marrow. After that, cells were seeded in methylcellulose (MethoCult M3534, StemCell Technology, Canada) at a concentration of 500 HSC per well in biological duplicates. The methylcellulose plates were incubated at 37°C for 12–15 days. For analysis, the averages of the number of colonies observed in the duplicates of each experimental condition were considered (Figure 8A).

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.