G csf

Primary cells were cultured in SFM supplemented with stem cell factor (SCF; 0.2 ng/ml; BioLegend, #573902), granulocyte colony-stimulating factor (G-CSF; 1 ng/ml; BioLegend, #578602), granulocyte-macrophage CSF (GM-CSF; 0.2 ng/ml; BioLegend, #572902), interleukin-6 (IL6; 1 ng/ml; BioLegend, #570802), macrophage inflammatory protein α (MIPα; 0.2 ng/ml; PeproTech, #300-08), leukemia inhibitory factor (LIF; 0.05 ng/ml; PeproTech, #300-05), [bovine serum albumin (BSA), insulin, and transferrin (BIT)] (20%; STEMCELL Technologies, #09500), low-density lipoprotein (40 μg/ml; Sigma-Aldrich, #L4646), 2-mercaptoethanol (0.1 mM; Invitrogen, #31350-010), and 1% penicillin/streptomycin (LifeTech, #15140-122) and resuspended in Iscove’s modified Dulbecco’s medium (LifeTech, #12440-053). The CML cell lines K562 and KCL22 (DSMZ) were cultured in RPMI 1640 medium (LifeTech, #11875-093) supplemented with 1% penicillin/streptomycin, 1% l-glutamine, and 10% (v/v) fetal calf serum. Both primary cells and cell lines were passaged every 2 to 3 days, maintained at a concentration of 2 × 10⁵ cells/ml at 37°C with 5% CO₂, and routinely tested for mycoplasma. ULK1 KO single-cell cloning was performed with serial dilution in a 96-well plate (100 μl per well). Puromycin selection (3 μg/ml) was performed after cell expansion, and Western blotting was performed to validate ULK1 KO.

Commercial antibodies for use in flow cytometry were purchased from BD Biosciences, Biolegend or eBioscience. Recombinant mouse IL-2 and α-IL-2 monoclonal antibody, clone JES6-5H4 (eBioscience). IL-2/α-IL-2 complex was generated by incubating 1.5 μg recombinant mouse IL-2 with 8 μg JES6-5H4 for 15 minutes at RT. TL1A-Ig was generated as described previously^{28 (link)}. The A20^luc/YFP cell line (derived from BALB/c mice) was a generous gift of Dr. Robert Negrin (Stanford University) and used for GVL experiments^{30 (link)}. Luciferin was purchased from Perkin Elmer; G-CSF (Biolegend) and AMD3100 (Sigma Aldrich).

To assess myeloid cell growth and differentiation in bulk liquid culture, human HSPC were maintained in Iscove’s Modified Dulbecco’s Medium (IMDM; Fisher Scientific, Loughborough, UK) supplemented with IL-3, SCF, G-CSF and GM-CSF (BioLegend, London, UK) at 5 ng/mL for 13 days. Transduced cultures were analyzed by flow cytometry for cell surface markers expression using a BD FACSCanto™II (Supplemental Materials and Methods) as previously described [12 , 14 (link)]. The gating strategy employed in these studies is shown in Supplemental Fig. S1. Granulocytic cells were defined as CD13^−/+CD36⁻, monocytic cells as CD13⁺CD36⁺, and erythroid cells as CD13⁻CD36⁺. Measurement of cell motility using the Transwell^® cell migration assay was performed on day 6 of culture using stromal cell-derived factor 1 (SDF-1) as a chemoattractant (Supplemental Materials and Methods). Morphology was assessed on day 17 of culture (Supplemental Materials and Methods) [13 (link)].