Wang et al. Journal of Experimental & Clinical Cancer Research 2010, 29:140 http://www.jeccr.com/content/29/1/140 Page 3 of 7 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 pro- tein levels and different clinical and pathological features 2 were explored using cross tabulation and Pearson’s x . P values less than 0.05 were selected. Results HBO1 protein level correlates positively with ERa 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. We found that HBO1 which was predominantly detected in nucleus (Figure 1B-C) was highly expressed in breast cancer tissues (Table 1) and sig- nificantly correlated with ERa (p < 0.001) and PR (p = 0.002) (Table 2). Further statistical analysis revealed HBO1 protein level correlated positively with histology grade in ERa positive tumors (p = 0.016) rather than ERa negative tumors (Table 2). For benign breast tissues, low HBO1 immunoreactivity was observed and epithelial cells dis- played a minimal granular staining (Figure 1A). Moder- ately and poorly differentiated breast cancer tissues showed intense HBO1 staining (Figure 1B-C). To further study the relationship between HBO1 and ERa, we exam- ined HBO1 expression level in several breast cancer cell lines by western blot, which showed that ERa positive breast cancer cell lines exhibited higher HBO1 protein than ERa negative breast cancer cell lines (Figure 1D). E2 induces HBO1 expression in breast cancer cells In order to further investigate the relationship between ERa and HBO1, we treated breast cancer cells with Figure 1 Expression of HBO1 in human breast cancer. (A) Immunohistochemical staining for HBO1 in benign breast epithelial tissues (magnification: A, 400×). (B) Moderately differentiated breast cancer tissues (brown staining) (magnification: B, 400×). (C) Poorly differentiated breast cancer tissues (brown staining) (magnification: C, 400×). (D) HBO1 protein level in several breast cancer cell lines based on the western blot results.