Expression levels and transcriptional activity of ERβ in BG-1. A.

<p>BG-1 cells were infected with Ad5 or Adβ adenoviruses and treated for 24h with control vehicle ethanol (Control) or E2 10⁻⁸M (E2). The expression of ERβ and rS9 reference gene was measured by real-time PCR. Results represent the mean ± SD of ERβ expression normalized by rS9 of 3 independent experiments. Measurements of Adβ and Adβ+E2 groups were compared by unpaired Student's <i>t</i> test. ** p<0.001. <b>B.</b> Proteins were extracted from cells infected in the same conditions as in A and treated for 0, 3, 6 or 24h with E2 10⁻⁸M (E2). Levels of ERα and ERβ were analyzed by western blot. Actin was used as a loading control. The upper band of ERβ blot labeled with a star corresponds to aspecific staining. <b>C.</b> Transcriptional activity of ERβ. BG-1 cells were infected with Ad5 or Adβ adenoviruses and transfected with ERE2-TK-Luc reporter along with β-galactosidase reporter. The cells were treated with ethanol as vehicle (Control) or E2 (10−8M) for 24h. Results show relative Luc activities (% of values of Ad5-infected cells without E2) ± SD after normalization with β-gal activity (3 independent experiments). Measurements of Ad5+E2 and Adβ+E2 groups were compared by unpaired Student's <i>t</i> test. * p<0.05. <b>D.</b> PEO14 cells were infected with Ad5, Adα, Adβ or the combination of Adα and Adβ adenovirus and transfected with ERE2-TK-LUC reporter along with β-galactosidase reporter. Cells were treated with control vehicle (Control) or E2 (10−8M) for 24h. Results show relative luciferase activities (% of values of Ad5 infected cells without E2) ± SD after normalization with β-gal activity (3 independent experiments). Measurements of Adα, Adβ and Adα+β groups were compared by unpaired Student's <i>t</i> test. *** p<0.001. NS: non significant. <b>E.</b> Proteins from PEO14 cells infected in the same conditions as in D and treated with vehicle ethanol (Control) or E2 10⁻⁸M (E2) for 3, 6 or 24h were analyzed by western blot with antibodies against ERα and ERβ. Actin was used as a loading control. The upper band labeled with a star in right panel corresponds to aspecific staining.