Histone deacetylase inhibitors down-regulate G-protein-coupled estrogen receptor and the GPER-antagonist G-15 inhibits proliferation in endometriotic cells  Patrick Imesch, M.D., Eleftherios P. Samartzis, M.D., Konstantin J. Dedes, M.D., Daniel Fink, M.D., André Fedier, Ph.D.  Fertility and Sterility  Volume 100, Issue 3, Pages 770-776 (September 2013) DOI: 10.1016/j.fertnstert.2013.05.008 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Effects of romidepsin and SAHA on estrogen receptor (ER)-α, ER-β, and G-protein coupled estrogen receptor (GPER) protein expression in 11z epithelial endometriotic cells by Western blot analysis. Cells were incubated with either romidepsin (A) or SAHA (B) at the concentrations and for the time periods indicated. Controls were treated with dimethyl sulfoxide (DMSO) alone in the highest corresponding concentration. Cells were then lysed, and proteins were separated by polyacrylamide gel electrophoresis and blotted. The respective complexes were detected by chemiluminescence and autoradiography. Tubulin was the sample loading control (representative of two independent data sets). Ac-H3, Ac-H4 = histone deacetylases. Fertility and Sterility 2013 100, 770-776DOI: (10.1016/j.fertnstert.2013.05.008) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effects of romidepsin and SAHA on G-protein coupled estrogen receptor (GPER) messenger RNA (mRNA) expression in 11z epithelial endometriotic cells by quantitative real-time polymerase chain reaction (PCR). Cells were incubated with either romidepsin (A) or SAHA (B) at the concentrations indicated for 24 and 48 hours. Controls were treated with dimethyl sulfoxide (DMSO) alone in the highest corresponding concentration. The data are presented as the mean ± SD of three independent experiments. Fertility and Sterility 2013 100, 770-776DOI: (10.1016/j.fertnstert.2013.05.008) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Effects of G-1 alone, G-15 alone, or a combination of G-1 and G-15 on Akt phosphorylation and cell proliferation in 11z epithelial endometriotic cells. (A) Akt phosphorylation was assessed by Western blot analysis in cells treated with G-1 (30 μM) alone for 2 hours, G-15 (60 μM) alone for 4 hours, or a combination of 60 μM G-15 and 30 μM G-1 (G-1 added 2 hours after G-15). The cells were lysed, and the proteins were separated by polyacrylamide gel electrophoresis and blotted. Total Akt protein and phospho-Akt complexes were detected by chemiluminescence and autoradiography. Total Akt was used as the sample loading control (representative of two independent data sets). Cell proliferation was assessed in different settings by 93-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (Sigma) (MTT) analysis (details, see Materials and Methods section). (B, C) Cells in medium containing 3% steroid hormone-depleted serum were left untreated or treated with either G-1 (3, 10, or 30 μM) or G-15 (30 or 100 μM) for 72 hours. (D) Cells in medium containing 3% steroid hormone-depleted serum were left untreated (none) or treated with 10 μM G-1 (G-1) or 30 μM G-15 (G-15) for 72 hours or pretreated with 10 μM G-1 for 72 hours followed by treatment with 30 μM G-15 for an additional 72 hours (G-1/G-15). (E) Cells in medium containing 0.1% steroid hormone-depleted serum were left untreated (none) or treated with 300 nM E2 (E2) or 3 μM G-15 (G-15) for 72 hours or pretreated with 3 μM G-15 for 2 hours followed by treatment with 300 nM E2 (E2) for an additional 72 hours (E2/G-15). (F) Cells in medium containing 10% fetal calf serum (FCS) were left untreated (control) or treated with 10, 30, or 60 μM G-15 for 24, 48, or 72 hours. The data are presented as relative proliferation in percentage of the control or effective optical density (OD) values as a function of treatment or time. Each experiment was independently performed at least twice in multiple cultures. The statistical significance of data points is indicated by asterisks, with P<.05 (compared to untreated controls). Fertility and Sterility 2013 100, 770-776DOI: (10.1016/j.fertnstert.2013.05.008) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions