Dulbecco s modified eagle s media

HEK293T cells (ATCC) were cultured in Dulbecco’s modified Eagle’s media (DMEM; Gibco-Life Technologies, Grand Island, NY, USA) with 4.5 g/L glucose (25 mM), 10% fetal bovine serum (FBS; Gibco-Life Technologies), 1% penicillin–streptomycin (Pen-Strep; Gibco-Life Technologies) and 5 μg/mL plasmocin (InvivoGen, San Diego, CA, USA) at 37 °C with 5% CO₂. For osmotic stress, HEK293T cells were exposed to 400 mM of sorbitol (Sigma, St. Louis, MO, USA) for 4 h. For recovery assays, cells were washed with PBS once and maintained in DMEM for 4 h. To induce proteasome inhibition, cells were incubated with 10 µM of MG132 for 4 h. For fluorescence in situ hybridization (FISH) and immunofluorescence, cells were seeded on coverslips coated with attachment factor protein (Gibco-Life Technologies). Cells on coverslips were fixed with 4% paraformaldehyde (PFA)-PBS (Electron Microscopy Sciences, Hatfield, PA, USA, 32%) solution for 10 min and washed with PBS.

MOLM-13 (DMSZ # ACC554), F36-P (DMSZ # ACC543) and SKK-1 have been obtained from DSMZ as a collaboration with Hans Drexler and have been characterized in detail^{36 (link)}. SKK-1 cells are available from the original authors^{37 (link)}. MDS-L cells were kindly provided by Tohyama and colleagues^{61 (link)}.
Cells were maintained in RPMI 1640 (Gibco, Thermo Scientific, Waltham, MA) supplemented with 10% of fetal bovine serum (FBS), 1% penicillin–streptomycin and 1% l-Gutamine (Gibco) at 37 °C in 5% CO₂. HEK293T (ATCC #CRL-11268) and HS-5 (ATCC #CRL-11882) were obtained from American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s media (Gibco) supplemented with 10% FBS, 1% penicillin–streptomycin and 1% l-Glutamine (Gibco) at 37 °C in 5% CO₂. Cells were authenticated and passaged for <6 months. CD34+ cells were isolated from mononuclear cells of healthy donors (see Supplementary Data 2 for donor characteristics) and cultured in suspension as described^{62 (link)}. For combinatorial treatments, cells were treated with AZA (Sigma-Aldrich, St. Louis, MO), decitabine (Sigma-Aldrich), C646 (Sigma-Aldrich), A-485 (Tocris, Bristol, UK), iCBP-112 (Tocris), CCS1477 (CellCentric, Ltd, Cambridge, UK), JQ-1 (Sigma-Aldrich), DMSO (Sigma-Aldrich) or CHX (Sigma-Aldrich) at the indicated amounts.

1-Deoxynojirimycin was purchased from Sigma-Aldrich (St Louis, MO, USA). QGreenTM 2x SybrGreen qPCR Master Mix was obtained from CellSafe (Suwon, Korea). Mouse hypothalamic GT1-7 cells were grown in Dulbecco's modified Eagle's media (Gibco, Rockville, MD, USA) and added with 10% heat-inactivated fetal bovine serum (FBS; Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco).

Primary cortical neurons were cultured from Swiss–Webster embryos (embryonic day 16), and then were maintained in neurobasal media (Gibco, 21103) supplemented with l-glutamine (5 mM), penicillin and streptomycin, and B27 neuronal additive. Primary astrocytes were cultured from Swiss–Webster pups (postnatal day 0), and then were maintained in Dulbecco’s modified Eagle’s media (Gibco, 10566) supplemented with l-glutamine (5 mM), penicillin and streptomycin, and fetal bovine serum. Cell viability was determined by the CellTiter-Glo® Luminescent Cell Viability Assay (Promega).

During the anterior cervical decompression surgery, 37 posterior longitudinal ligament specimens were obtained. To avoid contamination with osteoblasts or osteocytes, the ligaments were extracted carefully, cut up into 1-mm² pieces and washed with PBS several times. Subsequently, the specimens were divided into two parts. One was stored in liquid nitrogen for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. The remaining were washed with normal saline, plated in 35-mm culture dishes, and maintained in Dulbecco’s modified Eagle’s media supplemented with 10% fetal bovine serum (Gibco, USA). All assays were carried out on the fifth passage cell cultures.