Fig 2: Representative images of immunohistochemical staining for (A) STS, (B) SULT1E1 and (C) ERa subsequent in paraffin-embedded sections from serous epithelial ovarian cancer. Cytoplasmic staining was observed for STS and SULT1E1, while ERa staining was predominantly observed in the nucleus. Respective images from negative control samples for (D) STS, (E) SULT1E1 and (F) ERa. Magnification, ×200. STS, steroid sulfatase; SULT1E1, estrogen sulfotransferase; ERa, estrogen receptor a.

Fig 3: Hepatic SULT1E1 expression in Akita-CAR KO males. (a) Hepatic RNAs were prepared from C57BL/6J (N = 10) and Akita (N = 10) males. CYP2B10 mRNA, the classic CAR target showed the higher expression in Akita compared to C57BL/6J. Data set was analyzed by a Student’s t-test, and p-value is lower than 0.0001. (b) Nuclear extracts were separately prepared form livers of C57BL/6J (N = 3) and Akita (N = 3). The expression of CAR in nucleus was higher than that of C57BL/6J. (c) Hepatic RNAs were prepared from CAR WT (N = 9), CAR KO (N = 8), Akita (N = 9) and Akita-CAR KO (N = 6). Data set was analyzed by Kruskal-Wallis test, and p-value is 0.0001. (d) Chromatins were separately prepared from three livers of each of the above-mentioned groups for subsequent ChIP assays. Both of (e,f) presented the quantifying data, which were normalized by input intensity, for P-ERa and ERa respectively. The densitometry was performed by ImageJ. All data are presented as means ± S.D. of values of individual mice and one-way ANOVA was used as a statistical analysis. Each p-value of both groups is lower than 0.0001.

Fig 4: Intratumoral synthesis of E2 and its inactivation by sulfate conjugation from circulating steroid hormone precursors in cancer cells. E2 is synthesized from E1S in the sulfatase pathway, and from DHEA-S in the aromatase pathway. E1S and DHEA-S are taken up from the circulation by transporters, such as those from the OATP family encoded by SCLO. In the sulfatase pathway, HSD-17ß1 generates E2 from E1S, while E1 and E2 are inactivated by sulfonation by SULT1E1. Sulfonated estrogens do not bind to the ERa or ERß. Aromatase generates E2 from androgenic precursors via the aromatase pathway. E2, 17ß-estradiol; -S, sulfate; E1, estrone; STS, steroid sulfatase; SULT1E1, estrogen sulfotransferase; ER, estrogen receptor; DHEA, dehydroepiandrosterone sulfate; HSD-17ß, 17ß-hydroxysteroid dehydrogenase; OATP, organic anion transporting polypeptides; SLCO, solute carrier for organic anions.

Fig 5: Non-phosphorylated ERa is not bound to the Sult1e1 promoter in male mice with P-RORa. ChIP assays were applied to show RORa bindings to a proximal Sult1e1 promoter. (a) Chromatins were prepared from livers of ERa WT and KI males which were subjected to ChIP assays with either an RORa antibody or a P-S100 peptide antibody. Both of (b,c) indicate the data quantification, which was normalized by input intensity, for P-RORa and RORa respectively. The assay was examined by ImageJ, and one-way ANOVA were used as statistical analyses, and p-values are 0.0247 and 0.1080 respectively. (d) V5-CAR WT (no tag) was co-expressed with FLAG-tagged RORa WT, RORa S100D, EYFP-tagged ERa WT, and ERa S216D in Huh7 cells. Whole cell lysates were isolated and precipitated with aGFP-resin, and eluted proteins were loaded on a SDS-PAGE gel and subjected to staining with an aGFP- or aFLAG antibody.