Figure 5.

Contrary to ERβ5 and ERβ2, both ERβ1 and ERβ4 promote HGrC1 cell apoptosis. HGrC1 were transiently and efficiently (~70%) transfected by nucleofection with either Flag (C), Flag-ERβ1 (β1), Flag-ERβ2 (β2), Flag-ERβ4 (β4), or Flag-ERβ5 (β5). (A) MTT assay was performed 24 h and 48 h post-transfection. The quantity of formazan, which is directly proportional to the number of viable cells, was measured by recording changes in absorbance at 575 nm. Fold change in growth was expressed as the ratio of the OD_575nm value at 48 h post-transfection onto that measured at 24 h post-transfection. Contrary to Flag-ERβ5 (β5) or Flag-ERβ2 (β2), Flag-ERβ4 (β4) as well as Flag-ERβ1 (β1) significantly reduced the fold change in cell growth. Each experiment was performed 3-6 times, and data are reported as means ± SEM from four replicates. * p ≤ 0.05 vs. control Flag (C) by the Mann–Whitney test. (B) Twenty-four hours post-transfection, annexin V cell binding was assessed followed by measurement of the total cell number. Values represent the ratio between annexin V binding to the total cell number. Contrary to Flag-ERβ5 (β5) or Flag-ERβ2 (β2), Flag-ERβ4 (β4) as well as Flag-ERβ1 (β1) significantly increased cell apoptosis. Means ± SEM in four replicates performed 3–6 times * p ≤ 0.05 vs. control Flag (C) by the Mann–Whitney test.