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  • br incubated with an anti HA antibody in the presence


    incubated with an anti-HA antibody in the presence of protein G beads using a Protein G Immunoprecipitation kit (Sigma-Aldrich, Buchs, Switzerland) overnight at 4 °C. The beads were then washed three times and boiled for use in the western blot analysis. For the IP assay using A2780 cells, the Angiotensin II were lysed, and the supernatants were incubated with an anti-Bach1 or anti-HMGA2 antibody before the expression of Bach1 or HMGA2 was assessed by western blotting.
    2.11. Chromatin immunoprecipitation (ChIP) assay
    The ChIP assay was carried out as described previously [7]. In brief, cells were seeded into a 10 cm tissue culture dish, and one day later, they were cross-linked with 1% formaldehyde for 10 min at room temperature, followed by genomic DNA fragmentation using a sonica-tion apparatus. The chromatin fragments derived from A2780 cells were immunoprecipitated with 5 μg of an antibody against Bach1 (AF5776, R&D Systems, Minneapolis, MN, USA) or an antibody against HMGA2 (SAB2701959, Sigma-Aldrich, Buchs, Switzerland). DNA ex-traction was performed using a Qiagen Purification kit. Real-time PCR analysis was performed with primers amplifying the promoters of Vi-mentin, Slug, Snail and Twist. The primers are listed in Supplementary Table 3.
    2.12. Analysis of Slug promoter activity
    A luciferase assay was performed as described previously [7]. Briefly, A2780 cells were transfected with the Bach1-FLAG or control plasmid. Twenty-four hours later, the cells were transfected with a β-galactosidase plasmid and the wild-type or mutated versions (gaaag-gactaagcag→gaaaggactaGTAag) of the Slug promoter plasmid or the pGL3-basic luciferase reporter plasmid. Transfection was performed with Lipofectamine 2000, and the transfected cells were cultured for an additional 24 h; then, the cells were harvested, and luciferase activity was measured with a Luciferase Assay Kit (Promega, Madison, WI, USA). β-Galactosidase (β-gal) activity was measured. The relative Luc activity was calculated as the ratio of Luc/β-gal activity. Each experi-ment was performed independently three times.
    2.13. Analysis of TCGA data for human ovarian cancer
    2.14. Statistical analysis
    The data are presented as the means ± SEMs. Differences among groups were determined with One-way analysis of variance (ANOVA) with post hoc test for multiple comparisons. Differences between two groups were assayed by two-tailed Student's t-test using the SPSS 19.0 software package. Statistical significance was defined as P < 0.05.
    3. Results
    3.1. Bach1 is highly expressed in ovarian cancers and predicts poor prognosis
    By analyzing EOC and normal ovarian tissues, we found that Bach1 protein levels were low in normal ovarian tissues, relatively higher in
    Fig. 1. Bach1 is highly expressed in ovarian cancers and predicts poor prognosis. A. Representative images of Bach1 immunostaining. Bach1 expression was determined by immunostaining in 5 normal ovary samples, 21 samples of early-stage EOC (stage I or II), and 9 samples of advanced-stage EOC (stage III or IV). The stage of EOC was defined using the International Federation of Gynecology and Obstetrics (FIGO) staging systems. Scale bar, 200 μm. B. The IHC scores for Bach1 in EOC and normal ovarian tissues. ANOVA with post hoc test. C. Bach1 mRNA expression in healthy ovaries (n = 8) and ovarian cancers (n = 586) of stage I-IV was retrieved from the TCGA dataset (probe 204194_at). t-test. D-E. The progression-free survival (PFS) rates of 1435 patients and overall survival (OS) rates of 1656 patients with ovarian cancer were compared between the low-Bach1 and high-Bach1 groups based on extracted clinical data from the TCGA (http://tcga-data.nci.nih. gov).