Fig 2: Co-expression of ERa, ERß, and GPER in EC tissue cores of the tissue microarrays. Positions of samples in tissue microarrays are marked by letters A–D and numbers 1–6. The whole TMAs and control endometrial tissue (D2) and EC tissue (C2) cores are shown for ERa (anti-ERa antibodies, 1D5 Dako, Cat. #: M7047, lot 1: 00034057, lot 2: 20015818), ERß (anti-ERß antibodies, 14C8, GeneTex, Cat. #: GTX70174, Lot: 20882), and GPER (anti-GPER antibodies, HPA027052, Sigma-Aldrich, Cat. #:HPA027052, Lot: A61748) staining. The control staining was carried out without the primary antibodies. Sections were also stained with hematoxylin and eosin (HE). Scale bar, 50 µm.

Fig 3: ERß mRNA and protein levels in endometrial cancer and adjacent control endometrium. (A) Before-and-after graph shows the normalized expression levels of the ESR2 gene in control endometrial tissue (Control) and the corresponding EC tissue (Tumor). The levels of gene expression are on a logarithmic scale. (B) ECL detection of ERß (59 kDa band). 18 paired samples were analyzed using anti-ERß antibodies (ab3576, Abcam, Cat. #: ab3576, Lot: GR208064-1) and GAPDH was used as a normalization control. Before-and-after plots show quantification of Western blotting data. Below, representative membrane with ERß and GAPDH staining is shown. EC tissue (T), adjacent control endometrial tissue (C), placenta (P) was used as a control tissue. (C) IHC scores in 21 samples from adjacent control endometrial tissue (Control) and EC tissue (Tumor). Tables show mean scores ± standard deviations, while before-and-after graphs show nuclear and cytoplasmic ERß (anti-ERß antibodies, 14C8, GeneTex, Cat. #: GTX70174, Lot: 20882 (1:100). (D) IHC staining in representative paired adjacent control endometrial tissue (C38) and EC tissue (T38) for ERß. In the negative controls (Control), the primary antibodies were replaced with serum of the same animal species (rabbit, mouse). Anti-ERß antibodies were validated (Supplementary Figure S6 and Table S5).

Fig 4: Expression of ESR2 isoforms in endometrial cancer and adjacent control endometrium. (A) ESR2 isoforms a, g; (B) ESR2 isoforms b, d, h, k, and l; and (C) isoform f. Expression in 34 paired EC samples is shown.

Fig 5: ERa affects isoflavone-induced neurite outgrowth in neuronal-derived Neuro-2A cells. Cells were induced to differentiate through serum starvation for 1–3 days, followed by immunofluorescence analysis with ß-tubulin III (blue), doublecortin (red), and F-actin (green). (A) Representative photomicrographs showing the effects of isoflavones on the differentiation of Neuro-2A cells. (B) Changes in neurite lengths after exposure to isoflavones (n = 30). Quantitative analysis of the effect of isoflavones after exposure to ICI 182,780 (C), G15 (D) ERa siRNA (E), ERb siRNA (F), ERa and ERß siRNA (G), and GPER1 siRNA (H) on neurite length in Neuro-2A cells (n = 30). Bars indicate 50 µm. Data are shown as the mean ± standard error of the mean and represent at least three independent experiments. #### p < 0.0001 indicates statistical significance according to two-way or one-way analysis of variance, followed by the post hoc Tukey test compared to E2 (10 nM). **** p < 0.0001, *** p < 0.001, ** p < 0.01 and * p < 0.05 indicate statistical significance according to two-way or one-way analysis of variance, followed by the post hoc Tukey test.