Skno 1

The cell culture for leukemia cells was performed as our previous reports 28 (link). Human leukemia cell lines NB4, Kasumi-1, K562, U937, HL60, SKNO-1, MV4-11, MOLM13, MOLM14, and mouse C1498 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). NB4, K562, U937, HL60, MV4-11, MOLM13, and MOLM14 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum (FBS) (Life Technology, New York, USA). Kasumi-1 and SKNO-1 cells were cultured in RPMI1640 with 20% FBS. C1498 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) with 10% FBS.

The human AML cell lines (Kasumi-1, SKNO-1, and HL-60), were purchased from American Type Culture Collection (Manassas, VA, USA). The human ALL cell lines (Sup-B15, BV173, NALM-6, and BALL-1) were obtained from Guangzhou Jennio Biotech Co. Ltd (Guangzhou, China). The human AML cell line HEL, chronic myeloid leukemia cell line K562, the human lymphoma cell line Ramos and the human myeloma cell line KM-3 were kind gifts from Professor K. Y. Liu of Peking University People’s Hospital. These cell lines were maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 1% streptomycin and penicillin (all from Gibco, Billings, MT, USA) at 37 °C with 5% CO₂. Cell viability was observed daily with a microscope (Olympus, Ckx53sf, Tokyo, Japan), cell lines (except SKNO-1 cell line) were passaged every 2–3 days and SKNO-1 cells were passaged every 3-4 days. Asiatic Acid (Selleck Chemicals, Houston, TX, USA), was used as a P38 MAPK activator.

Kasumi-1, SKNO-1, U937, and K562 cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in Gibco RPMI-1640 (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with streptomycin (100 µg/mL, Gibco), penicillin (50 U/mL, Gibco), and 10% fetal bovine serum (FBS; Gibco). As previously described [13 (link)], SKNO-1-siAE cell line was generated by transfection with a lentiviral vector encoding siAGF1 oligonucleotides against the AML1-ETO mRNA fusion site to silence the expression of the AML1-ETO protein in SKNO-1 cells [17 (link)]. Cells were cultured in plastic tissue culture plates in a humidified 5% CO₂ atmosphere at 37 °C. SKNO-1-siAE cell were cultured for a month before sequencing. Lyophilized DAC (Topscience, Shanghai, China) and Ara-C (Topscience) were dissolved in dimethyl sulfoxide (Thermo Fisher Scientific) and stored at − 80 °C.

AML cell lines Kasumi-1 and SKNO-1 were obtained from American Type Culture Collection (ATCC, Manassas, VA). Peripheral blood mononuclear cells (PBMCs) enriched by Ficoll separation were obtained from leukemia patients and healthy donor. The protocols used in this study were approved by Rui Jin Hospital Ethics Review Boards. Written informed consents were obtained from all the patients and healthy donors in accordance with the Declaration of Helsinki. Animals were used according to the protocols approved by Rui Jin Hospital Animal Care and Use Committee. Kasumi-1 cells and PBMC were incubated in RPMI 1640 media (Gibco/Life Technologies, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotic/antimycotic (FBS; Hyclone Laboratories, Logan, UT) at 37°C in an atmosphere of 5% CO₂. FTY720 was purchased from Selleck. Caspase-3 specific inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-cho), neutral sphingomyelinase (nSMase) inhibitor GW4869, and protein phosphatase 2A (PP2A) inhibitor okadaic acid (OA) were purchased from Sigma-Aldrich. N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) was purchase from R&D System. Sphingolipid standards such as C14, C16, C18, C20, and C22 ceramides were available from Toronto Research Chemicals Inc and sphingosine, S1P were purchased from Sigma.

Kasumi-1 and SKNO-1 cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany), respectively. Cell were maintained in RPMI-1640 media supplemented with 20% FBS. SKNO-1 cells were also supplemented with 10ng/ml recombinant GM-CSF. Following antibodies were used in this study: RUNX1 (Cat. No. 4334, Cell Signaling Technology, Inc., Danvers, MA), ETO (Cat. No. 4498S, Cell Signaling Technology, Inc.), RUNX1T1/ETO (Cat. No. ab124269; Abcam, Cambridge, MA), CDK6 Clone DCS83 (Cat. No. 3136S; Cell Signaling Technology, Inc.), Tubulin Clone DM1A (Cat. No. T9026; Sigma-Aldrich, St. Louis, MO), and normal (Cat. No. sc-2027 for anti-rabbit; Cat. No. sc-2025 for anti-mouse) and HRP-conjugated IgG (Cat. No. sc-2004 for anti-rabbit; Cat. No. sc-2005 for anti-mouse) antibodies (Santa Cruz Biotechnology, Dallas, TX).

The t(8:21)-bearing cells lines, Kasumi-1 and SKNO-1, were obtained from the American Type Culture Collection (Manassas, VA, USA). These cells were cultured in Dulbecco’s RPMI 1640 (Gibco BRL, Grand Island, NY, USA) supplemented with 15% fetal bovine serum (Gibco BRL), 100 U/mL penicillin and 100 U/mL streptomycin as previously descried [35 (link)]. Normal human hematopoietic cells were isolated from peripheral blood collected from five volunteer healthy donors using Ficoll density gradient centrifugation. All cells were incubated in a 5% CO₂ incubator at 37 °C.