Horse serum

MDA-MB-231, Hs578T, BT20, MCF7, and H1299 cells were purchased from Korea Cell Line Bank (Seoul, Korea). MCF10A cells were obtained from the American Type Culture Collection (Manassas, VA, USA). MDA-MB-231, BT20 and MCF7 cells were maintained in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% (v/v) fetal bovine serum (FBS; Biowest, Nuaillè, France), 100 units/mL penicillin, and 100 μg/mL streptomycin (Welgene, Korea). Hs578T cells were maintained in Dulbecco’s Modified Eagle’s Medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% (v/v) FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin. MCF10A cells were cultured in DMEM/F12 medium (Invitrogen, Carlsbad, CA, USA) containing 10 μg/mL bovine insulin, 20 ng/mL epidermal growth factor, 100 ng/mL cholera enterotoxin, 0.5 μg/mL hydrocortisone (Sigma, St. Louis, MO, USA), and 5% horse serum (Welgene, Korea).

BMSCs were obtained and maintained from WT and KO mice^{56 (link)}. Briefly, bone marrow (BM) cells were isolated from the femurs and tibias of 8-week-old mice and cultured in Dulbecco’s modified Eagle medium (Welgene LM 001-05) supplemented with 10% fetal bovine serum (FBS; Welgene S 001–01), 10% horse serum (Welgene S 104–01), 2 mM L-glutamine (Welgene LS 002–01), 2 mM sodium pyruvate (Welgene LS 002–02), and 100 U ml⁻¹ penicillin/streptomycin (Welgene LS 202–02) at 37 °C and 5% CO₂. Non-adherent cells were removed by washing with PBS after overnight incubation. Adherent cells were cultured and fresh medium was added every 2 days. After 4 weeks of culture, cells were harvested by incubation with trypsin/EDTA (Welgene LS 015–01) for 2 min at 37 °C and plated onto culture dishes. At two weeks after the first passage, cells were harvested and cultured under the same conditions for subsequent passages (passages 2–7). OP9 BMSC line (ATCC CRL-2749) was maintained in alpha MEM (Welgene LM 008-02) supplemented with 20% FBS and 1% penicillin/streptomycin at 37 °C and 5% CO₂. For induction of VCAM-1, OP9 cells were treated with TNF-α (100 ng ml⁻¹; R&D Systems 410-MT) for the indicated time period. To inhibit Ezh activity, OP9 cells were treated with various concentrations of GSK126, an Ezh2 inhibitor (Xcessbio Biosciences M60071-2), for 48 h.

J558 and BCL1 strains were purchased from ATCC, and M12.4.1 cells (31 (link)) were provided by Dr. Mark Boothby (Vanderbilt University, Nashville, TN, USA). J558 cells were cultured with DMEM (Welgene, Daegu, South Korea) supplemented with 10% horse serum (Welgene) and penicillin/streptomycin (Welgene). One million cells were transfected with 5 μg DNA by electroporation using Neon transfection system (Thermo Fisher Scientific) or NEPA21 Super Electroporator (Nepa Gene). Transfection efficiency was about 10–20%. Sixteen hours after transfection, the cells were harvested and subjected to further experiments. Cell viability was measured by the trypan blue exclusion method. To detect cell death, cells were stained with 7-aminoactinomycin D (7-AAD) and Annexin V (BD Biosciences) and assayed by flow cytometry.