Wang et al. Journal of Experimental & Clinical Cancer Research 2010, 29:140 http://www.jeccr.com/content/29/1/140 Page 2 of 7 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 17b-estradiol (E2) were purchased from Sigma. Lipofectamine 2000, Tri- zol 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 pur- chased from Roche. RNA PCR Kit (AMV) Ver.3.0 was pur- chased from TaKaRa. Polinl-2-plus kit was a product of GBI. Anti-phospho-ERK1/2 (Thr202/Tyr204) and anti- ERK1/2 antibodies were purchased from Cell Signaling Technology. ERa siRNA, mouse monoclonal anti-ERa, anti-GAPDH, 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 pur- chased 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 supple- mented with 10% fetal calf serum (FBS), 10 ug/ml of insu- lin, 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 strep- tomycin. SiRNA was transfected with X-tremeGENE siRNA Transfection Reagent according to the manufac- ture’s instructions. Western blot analyses Proteins were detected by western blot analysis as described previously [10]. The cells were lysed by lysis buf- fer, separated in 10% SDS-PAGE and then transferred to a PVDF membrane. The membrane was incubated with pri- mary 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 paraf- fin-embedded for histopathologic diagnosis and immuno- histochemical 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. Sections were dewaxed in xylene and rehydrated in graded ethanol. Endogenous peroxidase activity was blocked by immer- sion in 0.3% methanolic peroxide for 30 minutes. Next, sections were microwaved in citrate buffer for antigen retrieval. Rabbit polyclonal anti-HBO1 (1:100 dilutions) was used as primary antibodies. Staining was com- pleted with Polink-2-plus kit, in accordance with the manufacturer’s instructions. Color reaction product was developed with diaminobenzidine. All sections were counterstained with hematoxylin. Two patholo- gists separately blinded to the clinical outcome of the patients evaluated all samples. The immunoreactivity intensity was evaluated as 0 (absent); 1 (weak nuclear staining); 2 (moderate nuclear staining of intermediate level); 3 (more coarse nuclear staining). Positive cells were quantified as a percentage of the total number of tumor cells with observation of 1000 cells in 5 high power field (×400), and assigned to one of five cate- gories: 0: < 5%, 1: 5-25%, 2: 26-50%, 3: 51-75% and 4: > 75%. HBO1 expression was scored semi-quantita- tively using the Remmele-score (immunoreactive score (IS) = score of percentage of positive tumor cells × score of staining intensity). Then the slide of IS > 3 was classified as a positive case [11]. Reproducibility of the scoring method used by both observers was greater than 95%. Total RNA extraction and real-time PCR Total mRNA samples of T47 D and MCF-7 breast cancer cells were extracted using trizol reagent according to the manufacturer’s instructions. One microgram of total RNA extracted from the cells was subjected to reverse transcription (RT). The RT and real-time PCR were per- formed by using TaKaRa RNA PCR Kit (AMV) Ver.3.0 and SYBR Premix Ex Taq II according to the manual respectively. Primers used for real-time PCR were as fol- lows: HBO1-F 5’-GATGCCCACTGTATCATAACC-3’ and HBO1-R 5’-TTCTTCCTGAGTTCAGCCACT-3’; GAPDH-F 5’-GGCTGAGAACGGGAAGCTTGTCAT-3’ and GAPDH-R 5’-CAGCCTTCTCCATGGTGGT- GAAGA-3’. Real-time PCR was performed using 7500 multicolor real-time PCR detection system (ABI) with the following cycling conditions: (i) 30s at 95°C and (ii) 40 cycles, with 1 cycle consisting of 5s at 95°C, 34s at