Chip chromatin immunoprecipitation kit

The enrichment of EZH2 in the promoter region of the FOXA1 gene was detected using a Chromatin Immunoprecipitation (ChIP) Kit (Millipore Corp.). When the cell confluence reached 70–80%, the cells were fixed in 1% formaldehyde for 10 min at room temperature to crosslink the intracellular DNA and proteins. Then, the crosslinked DNA and proteins were randomly sheared into appropriately sized fragments by an ultrasonicator for a total of 15 cycles (10 s each cycle at intervals of 10 s). The fragments were centrifuged at 30,237 × g at 4 °C; then, the supernatant was collected into three tubes, and the following antibodies were added: positive control antibody (RNA polymerase II), NC antibody (immunoglobulin G (IgG) of normal mouse), and rabbit anti-EZH2 antibody (ab228697, Abcam). The tubes were incubated at 4 °C overnight, and the endogenous DNA–protein complex was precipitated with protein agarose/sepharose. The supernatant was discarded after transient centrifugation, the nonspecific complex was washed, and the crosslinks were broken at 65 °C overnight. The DNA fragment was extracted, purified by phenol/chloroform, and then recycled. The binding of EZH2 and the FOXA1 promoter region was tested using specific primers for the FOXA1 gene promoter region.

Breast cancer cell lines MCF-7, T47D, SUM1315 and MDA-MB-231 were purchased from ATCC (Manassas, VA, USA) and cultured in DMEM (Hyclone, Logan, Utah, USA) supplemented with 10% FBS (Gibco, Los Angeles, CA, USA). Primary antibodies against JMJD2A (C37E5), E2F1 (3742), HDAC1 (5356) and HDAC3 (3949) were purchased from Cell Signaling Technology (Boston, MA, USA). Anti-estrogen receptor alpha (anti-ERα) (ab2746), anti-progesterone receptor (PR) (ab32085), anti-human epidermal growth factor receptor-2 (anti-HER2) (ab134182), anti-E2F4 (ab150360) and anti-ARHI (ab107051) primary antibodies were all obtained from Abcam (Cambridge, UK). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (sc-25778) was purchased from Santa Cruz (CA, USA). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA). Trichostatin A (TSA) was purchased from Sigma Co. (St Louis, MO, USA). The chromatin immunoprecipitation (ChIP) kit was purchased from Upstate (a part of Millipore, Billerica, MA, USA). For the co-immunoprecipitation (Co-IP) assay, the protein G agarose beads and NP-40 lysis buffer were purchased from Beyotime Institute of Biotechnology (Nantong, China). Matrigel was purchased from BD Biosciences (San Jose, CA, USA). Cell counting kit-8 (cck-8) was purchased from Dojindo (Japan). And dual-luciferase reporter gene assay were from Promega (Madison, WI, USA).