Lp30 cells were generated by serial BMT/expansion of BM cells from leukemic primary CebpaLp30 mice. A pool of BM cells from tertiary expansions were used for both in vitro and in vivo experiments. BM cells were harvested from femur, tibia, and iliac bones; crushed; washed; pooled; and frozen in vials.

For in vitro proliferation, cell cycle, and apoptosis assays, Lp30 cells (female) were grown in R20/20 media: RPMI 1640 medium (Life Technologies) supplemented with 20% WEHI conditioned media, 20% fetal bovine serum (FBS), 1% penicillin/streptomycin (Pen/Strep), and cytokines (PeproTech): stem cell factor (SCF) (20 ng/ml) and hIL-6 (10 ng/ml). A2AR inhibitor (SCH58264, Sigma-Aldrich) was added from stock (5 mg/ml) in dimethyl sulfoxide (DMSO) to 30, 10, or 3 μM, and DMSO volume was normalized/added as mock.

BM cells from recipients transplanted with donor BM cells from Cbfb-MYH11 and KIT D816 mutant mice [Cbfb+/56m; Tg(Mx1-Cre)/KITD816Y/V] [referred to as Inv(16) cells] (47) were grown in x-vivo complete media: X-VIVO 15 with gentamicin (Lonza), supplemented with 10% bovine serum albumin (BSA) (Stem Cell Technologies), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), 1% l-glutamine (Gibco), 1% Pen/Strep, and cytokines (PeproTech): mSCF (50 ng/ml), hIL-6 (50 ng/ml), mIL-3 (10 ng/ml), and GM-CSF (10 ng/ml). Phoenix and HEK293T (human embryonic kidney line, female) cells were cultured in DMEM (Life Technologies) + 10% FBS and 1% Pen/Strep.

All cell cultures were mycoplasma-tested before freezing and never kept in culture for more than 3 months after rethawing. Phoenix and HEK293T cells were the only commercially available cell lines used, and virus production served as authentication of these cell lines.

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