Figure 4.

Absence of influence of ERβ2/β4/β5 on E2-induced ERβ1 transactivation in HGrC1-transfected cells. (A) The presence of ERα and ERβ (1, 2, 4, and 5) isoforms mRNA in hGCs (n = 49) and HGrC1 cells (n = 6) was evaluated by RT-qPCR (expression normalized to GAPDH). Values are represented as means ± SEM from three identical wells per patient, measured in triplicate. * p ≤ 0.05; ** p ≤ 0.01 by the Mann–Whitney test. A.U., Arbitrary Units. HGrC1 cells express low levels of endogenous ERα and ERβ isoforms, when compared to those expressed in hGCs. (B) HGrC1 cells were transiently cotransfected with the control Flag vector (C) alone or with the Flag-ERβ1 vector (β1), together with the Firefly luciferase reporter plasmid containing or not (pGL2) estrogen-response elements (EREs) upstream of luciferase (ERE-Luc). Flag-ERβ1 was also cotransfected (1:1 ratio) with either Flag-ERβ2 (β1+β2), Flag-ERβ4 (β1+β4), or Flag-ERβ5 (β1+β5). Renilla luciferase reporter plasmid was included as a normalizing transfection control. Twenty-four hours after solvent vehicle (V) or 10 nM E2 treatment, cells were lysed and analyzed for luciferase activity. Firefly luciferase activity was normalized to that of Renilla luciferase and reported as relative transactivation activities arbitrarily set at 1 for the control (C) solvent vehicle (V) condition in ERE-Luc-transfected cells. Each point represents the mean ± SEM in six replicates performed four times. * p ≤ 0.05; ** p ≤ 0.001 vs. vehicle (V) for each group of cotransfection, and ## p ≤ 0.001 vs. control (C) vehicle (V) using the Mann–Whitney test. A.U., Arbitrary Units. (C) HGrC1 cells were transiently transfected with either Flag-ERβ1 (β1), Flag-ERβ2 (β2), Flag-ERβ4 (β4), or Flag-ERβ5 (β5). Twenty-four hours post-transfection, cells were fixed, permeabilized, and the localization of Flag-ERβ proteins was monitored by immunofluorescence using an anti-Flag antibody (green). Nuclei were stained with DAPI (blue). Representative images at 20x magnification are presented.