Endogenous peroxidase activity was blocked by immersion 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 completed with Polink-2-plus kit, in accordance with the manufacturer’s instructions. Color reaction product was developed with diaminobenzidine. All MGCD0103 molecular weight sections were counterstained with hematoxylin. Two

pathologists 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 Pritelivir cell line 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 categories: 0: < 5%, 1: 5-25%, 2: 26-50%, 3: 51-75% and 4: > 75%. HBO1 expression was scored semi-quantitatively 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 performed by using TaKaRa RNA PCR Kit (AMV) Ver.3.0 and SYBR Metalloexopeptidase Premix Ex Taq II according to the manual respectively. Primers used for real-time PCR were as follows: HBO1-F 5′-GATGCCCACTGTATCATAACC-3′ and HBO1-R 5′-TTCTTCCTGAGTTCAGCCACT-3′; GAPDH-F 5′-GGCTGAGAACGGGAAGCTTGTCAT-3′ and GAPDH-R 5′-CAGCCTTCTCCATGGTGGTGAAGA-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 60°C. GAPDH was employed as an internal control under the same experimental conditions. Data were analyzed by using 7500 software (ABI). The values were obtained through normalizing to GAPDH copies. Statistical analysis Statistical data analyses were performed using SPSS 11.5 statistical software package. The relationship between check details protein levels and different clinical and pathological features were explored using cross tabulation and Pearson’s x2. P values less than 0.05 were selected. Results HBO1 protein level correlates positively with ERα In order to explore the biological role of HBO1 in human breast cancer in vivo, we examined the expression of HBO1 in tumor samples of primary breast cancer (n = 112) by IHC analysis.