Stem cell factor (scf)

BALB/c mice from both sexes were used. Animals were obtained from the Oswaldo Cruz Foundation breeding unit (Rio de Janeiro, Brazil). The protocols were approved by both Federal University of Rio de Janeiro Animal Use and Oswaldo Cruz Foundation Animal Welfare Committees. Eosinophils were differentiated in vitro from mouse bone marrow cells as previously described (25 (link)). Briefly, bone marrow cells were collected from femurs and tibiae of wild-type BALB/c mice with RPMI 1640 containing 20% FBS. Cells were cultured at 10⁶ cells/mL in RPMI 1640 containing 20% FBS (VitroCell), 100 U/mL penicillin, 10 μg/ml streptomycin, 2 mM glutamine and 1 mM sodium pyruvate (Sigma), 100 ng/mL stem cell factor (SCF; PeproTech) and 100 ng/mL FLT3 ligand (PeproTech) from days 0 to 4. On day 4, SCF and FLT3-L were replaced with IL-5 (10 ng/mL; Peprotech). On day 14, eosinophils were enumerated (purity ≥ 90%).

Neutrophils were collected from bone marrow cells of female C57BL/6J mice as previously reported [26 (link)]. Bone marrow cells were suspended in RPMI 1640 medium containing 2% NCS and carefully added onto 62% Percoll (GE Healthcare, Chicago, IL, USA) in RPMI 1640 medium. After centrifugation at 1000× g for 20 min at 25 °C, neutrophils were precipitated.
Eosinophils were prepared by differentiation from mouse bone marrow cells as previously reported [27 (link)]. Briefly, mouse bone marrow cells were cultured at 1.0 × 106 cells/mL in medium containing RPMI 1640 with 20% FBS, 100 IU/mL penicillin plus 10 μg/mL streptomycin (Nacalai Tesque), 25 mM HEPES (Nacalai Tesque), 1× nonessential amino acids (Nacalai Tesque), 1 mM sodium pyruvate (Nacalai Tesque) and 50 μM 2-ME (Thermo Fisher Scientific). From day 0 through 4, 100 ng/mL stem cell factor (SCF; [PeproTech]) and 100 ng/mL FLT3 ligand (PeproTech) were supplemented. On day 4, the medium with SCF and FLT3 ligand was replaced to the medium containing 10 ng/mL mouse recombinant IL-5 (Peprotech). From day 8 through 10, the cells were transferred to a new flask and the medium was changed. The cell concentration was adjusted to 1.0 × 106 cells/mL every day. On day 12, eosinophil differentiation was assessed by FACS staining with BV421-anti-Siglec-F and the cells were used for experiments between day 12 and day 19.

Bone marrow cultures were performed using a modified protocol of [68 (link)]. Briefly, WT and Foxo3^-/- bone marrow cells were enriched for hematopoietic progenitors using the EasySep Mouse Hematopoietic Progenitor Cell Enrichment Kit (#19756 Stemcell Technologies) and expanded at < 2x10⁶ cells/ml in non-treated 24-well plates with erythroid expansion medium. Erythroid expansion media consists of Stem Span SFEM (StemCell Technologies) supplemented with 2 U/ml human recombinant EPO (Amgen), 100 ng/ml SCF (PreproTech), 40 ng/ml insulin-like growth factor–1 (PreproTech), 10⁻⁶ M dexamethasone (D2915; Sigma), 0.4% cholesterol mix (Gibco) and 1% penicillin/streptomycin (Gibco). Two days later, cells were washed twice with PBS and plated at a concentration <1x10⁶ cells/mL in erythroid differentiation medium consisting of IMDM supplemented with 2 U/ml EPO, 100 ng/ml SCF, 10% Serum replacement (Invitrogen), 5% Platelet-Derived Serum, glutamine, MTG (1.27 μl in 10 ml of 1:10 dilution) and 10% Protein-Free Hybridoma Media. Cells were incubated in maturation medium for 3 days, with additional media added at day 3.

FACS-sorted LSK⁺ cells (5 000 cells per well in 96-well round-bottomed plates) were cultured in X-VIVO-15 medium (Lonza) containing 3% BSA, 100 U·mL⁻¹ penicillin, and 100 μg·mL⁻¹ streptomycin. For myeloid differentiation, a cytokine cocktail including 20 ng·mL⁻¹ IL-3, 20 ng·mL⁻¹ IL-6, 50 ng·mL⁻¹ SCF and 30 ng·mL⁻¹ M-CSF (Peprotech) was used. The lymphoid differentiation cytokine cocktail contained 100 ng·mL⁻¹ IL-7, 50 ng·mL⁻¹ Fit3L and 50 ng·mL⁻¹ SCF (Peprotech). Half of the cytokine-containing culture medium was refreshed every other day. The cells were cultured for a total of 7 or 12 days for myeloid or lymphoid cell differentiation, respectively.

The hiPSCs were differentiated into HPCs using the embryoid bodied (EB)-based HPC differentiation method [32 (link),33 (link)]. hiPSCs were briefly treated with the indicated flavonoids for 3 days before differentiation into EBs. For EB formation, cells were transferred onto Corning Ultra-Low Attachment Surface dishes with mTesR1 supplemented with 10 μM ROCK inhibitor for 6 days. On day 7, the formed EBs were transferred onto Matrigel-coated plate and incubated with the HPC differentiation medium (Iscove’s Modified Dulbecco′s Medium (Thermo Fisher Scientific) containing 20% FBS, with 100 ng/mL SCF (PeproTech), 10 ng/mL IL-3 (PeproTech), 10 ng/mL IL-6 (PeproTech), 20 ng/mL FLT3L (PeproTech), and 20 ng/mL BMP4 (PeproTech) with media exchange every 2 days until day 21. To obtain NKCs, hiPSC-derived HPCs were differentiated for 4 weeks in the presence of 10 ng/mL IL-15 (PeproTech), 5 ng/mL IL-3, 20 ng/mL IL-7 (PeproTech), 20 ng/mL SCF, and 10 ng/mL FLT3L. Medium containing cytokines was changed weekly with the exception of IL-3, which was only included for the first week of differentiation.

For cell lines and their driver oncogenes, see Supplementary Table S1. BV173, BV173R, 697, and SD1 cells were cultured in RPMI 1640 medium supplemented with 20% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Life Technologies, Paisley, UK). DOHH2, NALM6, REH, and SEM cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. SUP-B15 and SUP-B15R cells were cultured in McCoy's 5A medium (Life Technologies) supplemented with 20% FBS and 1% penicillin/streptomycin. RS4;11 cells cultured in α-MEM medium (Life Technologies) supplemented with 10% FBS and 1% penicillin/streptomycin.
Primary CD34⁺ ALL and normal cells were cultured in IMDM medium (Life Technologies) supplemented with 25% BIT (bovine serum albumin/insulin/transferrin; StemCell Technologies, Vancouver, BC, Canada), L-glutamine, penicillin/streptomycin, and 125 μM 2-mercaptoethanol (Life Technologies). ALL CD34⁺ cells were cultured in the presence of FLT3-ligand (100 ng/ml), IL-7 (100 ng/ml), and SCF (100 ng/ml), as described.^{25 (link)} Normal CD34⁺ cells were cultured in a five growth factor cocktail containing FLT3-ligand (100 ng/ml), G-CSF (20 ng/ml), IL-3 (20 ng/ml), IL-6 (20 ng/ml), and SCF (100 ng/ml) (all growth factors are from PeproTech, Rocky Hill, NJ, USA).