Interleukin 3 (il 3)

Immortalization of myeloid hematopoietic progenitors was performed by transduction of CD117 (c-Kit) positive bone marrow cells with a retrovirus expressing HoxB8 fused to the estrogen receptor^{21 (link)}. Cells were grown in 10 ng/ml IL-3 (Goldbio) and 1 μM estradiol (E2) (Sigma Aldrich). AML cell lines were obtained from ATCC between 2009–2014. They were validated by STR profiling in 2019 and undergo mycoplasma testing every 6 months. Myeloid progenitors, Nomo1, and U937 cell lines were grown in RPMI 1640 supplemented with 10% fetal bovine serum (Gibco, 10438026), 1% penicillin/streptomycin (Gibco, 15140122), and 1% GlutaMAX (Gibco, 35050061). 293T packaging cells were grown in DMEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% GlutaMAX. Proliferation was measured at the indicated times starting with a concentration of 10,000 cells/ml in the case of the immortalized murine myeloid progenitors and with 100,000 cells/ml for human cell lines. Cell number was determined by a hemocytometer counting chamber.

Wild-type or HMGN1-OE cells stably infected with the oncogenes indicated were seeded in methylcellulose media (Methocult M3234, Stem Cell Technologies), supplemented with IL-6 (10 ng/ml), SCF (10 ng/ml), and IL-3 (6 ng/ml) (GoldBio) at 2 × 10⁵ cells/ml and at 5 × 10⁴ cells/ml in subsequent passages. Colonies were manually counted at 7 days, pooled, and replated. For long-term culture initiating colony (LTC-IC) assays, leukemic splenocytes from three different animals were plated on MS5 feeder cells for three weeks in RPMI media enriched with IL-6 (10 ng/ml), SCF (10 ng/ml), and IL-3 (6 ng/ml). Cells were counted and 10,000, 20,000, or 50,000 GFP+ cells were seeded in methylcellulose media (Methocult M3234) supplemented with SCF (10 ng/ml), IL-3 (6 ng/ml), and IL-6 (10 ng/ml) in 35 mm dishes. One week later, the number of colonies and cells was determined.