The role of the AT1 receptor in 17β-oestradiol and EGF-mediated cell proliferation and Raf phosphorylation in breast cancer cells. (a) SKBR3 and MCF-7 breast cancer cells pretreated with or without angiotensin II receptor antagonist saralasin (10-6 mol/l) 1 hour before 24 hours of incubation with EGF (10 ng/ml) and 17β-oestradiol (10-8 mol/l) alone and in combination. Cell proliferation assays were carried out using MTT thiozolyl blue. Proliferative index of the control group is standardized to 1. The results shown are expressed as mean ± standard error (n = 9). Statistical analysis was performed using Mann-Whitney U test (*P < 0.02 versus without saralasin). (b) Western blot analysis of phospho-Raf and total Raf in SKBR3 cells pretreated with or without the AT1 antagonist saralasin (10-6 mol/l) for 1 hour before 10 minutes of incubation with EGF (10 ng/ml) and 17β-oestradiol (10-8 mol/l) alone and in combination. Results are representative of those obtained in three separate experiments. (c) Western bolt analysis of AT1 and phospho-Raf in SKRB3 cells transiently transfected with GAPDH siRNA, scrambled siRNA, or AT1 (#1 or #2) siRNA before treatment with 17β-oestradiol (10-8 mol/l) for 10 minutes. Results are representative of those obtained in three separate experiments. Optical density readings of control values were normalized to 1 and experimental groups were expressed as a ratio. Values are expressed as mean ± standard error (n = 3). (d) Immunohistochemistry was carried out for AT1 receptor on primary human breast cancer tissue (7 μm). Negative control was matched IgG. The slides were counterstained with Mayer's haematoxylin solution and were viewed under a light microscope at 40× and 200× magnifications. Positive cells stained cells stained brown against a blue background. (e) Immunofluorescence studies of AT1 receptor in human primary tumour breast cancer tissue (7 μm) and SKBR3 breast cancer cells. Slides were viewed under a confocal microscope at 630×. AT1, angiotensin II type 1; E2, 17β-oestradiol; EGFR, epidermal growth factor receptor; siRNA, small interfering RNA.