We found that HBO1 was notably increased in breast cancer. E2-upregulated HBO1 expression could be inhibited by ICI 182,780 or ERα RNAi in breast cancer cells. Furthermore, we also showed that ERK1/2 signaling pathway was involved in the expression of HBO1 increased by E2. Methods Materials Dulbecco’s modified Eagle’s medium (DMEM), phenol red-free DMEM (PR-free DMEM), U0126 and

17β-estradiol (E2) were purchased from Sigma. Lipofectamine 2000, Trizol Reagent and fetal bovine serum (FBS) were purchased from Invitrogen. Charcoal-stripped fetal bovine serum (CS-FBS) was purchased from Biowest. PVDF membrane, leupeptin, aprotinin, phenylmethylsulfonyl fluoride (PMSF) and X-tremeGENE siRNA Transfection Reagent were purchased from Roche. RNA PCR Kit (AMV) Ver.3.0 was purchased from TaKaRa. Polinl-2-plus kit was a product of GBI. Anti-phospho-ERK1/2 (Thr202/Tyr204) and #selleck kinase inhibitor randurls[1|1|,|CHEM1|]# anti-ERK1/2 antibodies were purchased from Cell Signaling Technology. ERα siRNA, mouse monoclonal anti-ERα, anti-GAPDH, selleck chemicals llc horseradish peroxidase (HRP)-conjugated goat anti-rabbit and HRP-conjugated goat anti-mouse IgG secondary antibodies were from Santa Cruz Biotechnology. Rabbit polyclonal

anti-HBO1 antibody (Catalog No: 13751-1-AP) was purchased from Protein Tech Group, Inc. The enhanced chemiluminescence (ECL) assay kit was purchased from Tiangen. Dual-luciferase reporter assay system was bought from Promega. ICI 182,780 was purchased from Tocris Bioscience. Cell culture and transfection Human MCF-7, T47 D, MDA-MB-453 and MDA-MB-435 breast cancer cells were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences. MCF-7 cells were cultured in DMEM supplemented with 10% fetal calf serum (FBS), 10 ug/ml of insulin, 100 U/ml of penicillin and 50 ug/ml of streptomycin. T47 D cells were maintained in DMEM supplemented with 10% FBS, 100 U/ml of penicillin and 50 ug/ml streptomycin. SiRNA was transfected with X-tremeGENE siRNA Transfection Reagent according to the manufacture’s instructions. Western blot analyses Proteins were detected

by western blot analysis as described previously [10]. The cells were STK38 lysed by lysis buffer, separated in 10% SDS-PAGE and then transferred to a PVDF membrane. The membrane was incubated with primary antibody followed by incubation with horseradish peroxidase-conjugated secondary antibody. Then the membrane was developed using the ECL detection system. Tissue samples 112 primary breast cancer specimens were consecutively obtained from pathological archives of Huashan Hospital, Fudan University from 2005 to 2008. There was no any bias for selection. Tissues were formalin-fixed and paraffin-embedded for histopathologic diagnosis and immunohistochemical study. The malignancy degree of tumor was scored according to the Scarff-Bloom-Richardson system. Immunohistochemistry (IHC) Serial sections (5 μm thick) were mounted on glass slides coated with 10% polylysine.