Mda mb 231

Four human breast cancer cell lines with different immunoprofiles, namely MDA-MB-361 (ER⁺ HER2⁺), MCF-7 (ER⁺ HER2^-), SK-BR-3 (ER^- HER2⁺), and MDA-MB-231 (ER^- HER2^-) were obtained from American Type Culture Collection (ATCC, USA) and were grown in culture flasks in HuMEC Basal Serum-free Medium (Thermo Fisher, USA) in order to prevent the influence of hormones and/or growth factors in regular serum-containing medium. For ER-positive cell lines (MDA-MB-361 and MCF-7), the cells were seeded in 96-well culture plates and incubated with various concentrations of AS extract, 17β-estradiol (0.1 µM, Sigma, USA), or the combination of 17β-estradiol (0.1 µM) and AS extract in medium for 48 h. For ER-negative cell lines (SK-BR-3 and MDA-MB-231), the cells were seeded in 96-well culture plates and treated with various concentrations of AS extract for 48 h. The cell viability and proliferative responses were determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and bromodeoxyuridine (BrdU) cell proliferation enzyme-linked immunosorbent assay (ELISA) (Roche, USA), respectively.

Human cell lines BT549, MDA-MB231, MCF-7, MDA-MB157, HCC70, A549 and NB4 were obtained from the American Type Culture Collection (ATCC) and cultured using ATCC recommended conditions. Primary antibodies against H3K79me2, H3K27me3, H3K4me3, H3K36me2, H4K20me3, H3K9me-pan were purchased from Cell Signaling (Danvers, MA). siRNA experiments were performed by transfecting MDA-MB-231 cells with X-tremeGENE Transfection Reagent (Roche) and 10 nM siRNA (Invitrogen), according to the manufacturer's protocol. The sequences for DOT1L siRNA (Invitrogen) were:

siRNA-1: 634-CGCGAGUUCAGGAAGUGGAUGAAAU;

siRNA-2: 117-CGAUAAACAUCACGAUGCUGCUCAU;

siRNA-3: 104-CGCUGCCGGUCUACGAUAAACAUCA;

siRNA-4: 470-CCCAGAUGAUUGAUGAGAUCAAGAU;

siRNA-5: 120-UAAACAUCACGAUGCUGCUCAUGAA.

siRNA-`mock represents an siRNA that has no homology to any vertebrate transcriptome.