Fig. 1 UGT2B15 mRNA levels are stimulated by E2 in a time- and dose-dependent manner in MCF-7 breast cancer cells. A, Time course for E2 treatment. Cells were treated with E2 (10 nm) for 2, 4, 8, 24, or 48 h. B, Dose response for E2 treatment. MCF-7 cells were treated with various concentrations of E2 (1 pm to 10 nm) for 8 h. RNA isolation, RT, and real-time PCR were performed as described in Materials and Methods. Values are the mean ± sem of three experiments. From: Estrogen Regulation of the Glucuronidation Enzyme UGT2B15 in Estrogen Receptor-Positive Breast Cancer Cells Endocrinology. 2006;147(8):3843-3850. doi:10.1210/en.2006-0358 Endocrinology | Copyright © 2006 by The Endocrine Society

Fig. 2 E2-induced increase in UGT2B15 mRNA levels is inhibited by ICI but not by CHX. A, MCF-7 cells were treated with ethanol vehicle, E2 (10 nm), ICI (1 μm), or a combination of E2 and ICI for 8 h. B, Cells were treated with ethanol vehicle (control), E2 (10 nm), PPT (100 nm), or genistein (Gen; 1 μm) alone or in combination with CHX (10 μm). RNA isolation, RT, and real-time PCR were performed as described in Materials and Methods. Values are the mean + sem of three experiments. From: Estrogen Regulation of the Glucuronidation Enzyme UGT2B15 in Estrogen Receptor-Positive Breast Cancer Cells Endocrinology. 2006;147(8):3843-3850. doi:10.1210/en.2006-0358 Endocrinology | Copyright © 2006 by The Endocrine Society

Fig. 3 UGT2B15 regulation by steroid hormone receptor ligands Fig. 3 UGT2B15 regulation by steroid hormone receptor ligands. A, Cells were treated with ethanol vehicle (control), 5α-DHT (10 nm), hydrocortisone (HC; 10 nm), R5020 (10 nm), or E2 (10 nm) for 8 h. B, Time course for E2 or DHT treatment. Cells were treated with E2 (10 nm) or DHT (10 nm) for 2, 4, 8, 24, and 48 h. C, Effect of antiestrogen (1 μm ICI) or antiandrogen (1 μm HFLT) on the stimulation of UGT2B15 mRNA by E2 (10 nm) or DHT (10 nm). Cells were treated with vehicle or ligand alone, or with ligand plus ICI or HFLT for 8 h. RNA isolation, RT, and real-time PCR were performed as described in Materials and Methods. From: Estrogen Regulation of the Glucuronidation Enzyme UGT2B15 in Estrogen Receptor-Positive Breast Cancer Cells Endocrinology. 2006;147(8):3843-3850. doi:10.1210/en.2006-0358 Endocrinology | Copyright © 2006 by The Endocrine Society

Fig. 4 UGT2B15 is an estrogen-regulated gene in several ER-positive human breast cancer cell lines. BT474, T47D, and ZR-75 cells were treated with E2 (10 nm) for 2, 4, 8, 24, or 48 h. RNA isolation, RT, and real-time PCR were performed as described in Materials and Methods. From: Estrogen Regulation of the Glucuronidation Enzyme UGT2B15 in Estrogen Receptor-Positive Breast Cancer Cells Endocrinology. 2006;147(8):3843-3850. doi:10.1210/en.2006-0358 Endocrinology | Copyright © 2006 by The Endocrine Society

Fig. 5 UGT2B15 is the only UGT2B enzyme regulated by estrogen in MCF-7 cells. Cells were treated with E2 (10 nm) for up to 48 h. RNA isolation, RT, and real-time PCR were performed as described in Materials and Methods. UGT2B4 and UGT2B7 mRNA were not detectable in MCF-7 cells using real-time PCR. From: Estrogen Regulation of the Glucuronidation Enzyme UGT2B15 in Estrogen Receptor-Positive Breast Cancer Cells Endocrinology. 2006;147(8):3843-3850. doi:10.1210/en.2006-0358 Endocrinology | Copyright © 2006 by The Endocrine Society

Fig. 6 UGT2B15 protein levels increase after E2 treatment in MCF-7 breast cancer cells. A, ELISA antibody titration assay. Preimmune (solid line) or immune (dashed line) antisera were incubated with BSA-conjugated antigen, and amount of antibody bound was determined by performing a colorimetric assay as described in Materials and Methods. Similar curves were obtained for immune antisera made to peptides 1 or 2. B, Cells were treated for 4, 8, 24, 48, or 72 h with E2 (10 nm). The blot was probed with anti-UGT2B15 antibody to peptide 2. β-Actin protein levels were also monitored to assess protein loading. Similar findings were observed in several repeat studies. From: Estrogen Regulation of the Glucuronidation Enzyme UGT2B15 in Estrogen Receptor-Positive Breast Cancer Cells Endocrinology. 2006;147(8):3843-3850. doi:10.1210/en.2006-0358 Endocrinology | Copyright © 2006 by The Endocrine Society

Fig. 7 Immunocytochemistry showing the increase in UGT2B15 protein in MCF-7 cells treated with E2. Cells were treated with vehicle for 72 h (B) or with E2 (10 nm) for 48 (C) or 72 (D) h. After treatment, cells were fixed and processed for immunocytochemistry as described in Materials and Methods. To demonstrate specificity of staining, a no primary antibody control (A) was performed with cells treated for 72 h with E2 (10 nm). Magnification, ×40. For the immunocytochemistry shown, antibody against peptide 1 was used, but similar results were obtained using antibodies against peptides 2 and 3. From: Estrogen Regulation of the Glucuronidation Enzyme UGT2B15 in Estrogen Receptor-Positive Breast Cancer Cells Endocrinology. 2006;147(8):3843-3850. doi:10.1210/en.2006-0358 Endocrinology | Copyright © 2006 by The Endocrine Society

Fig. 8 Assessment of UGT2B15 glucuronidation enzymatic activity of MCF-7 cells after E2 treatment. [C-14]Glucuronidated DHT was quantitated using microsomal fractions of vehicle or 10 nm E2-treated (for 72 h) MCF-7 cells after TLC and ImageQuant analysis. Values are mean ± sem of three experiments. From: Estrogen Regulation of the Glucuronidation Enzyme UGT2B15 in Estrogen Receptor-Positive Breast Cancer Cells Endocrinology. 2006;147(8):3843-3850. doi:10.1210/en.2006-0358 Endocrinology | Copyright © 2006 by The Endocrine Society