1. Differential expression of G-protein-coupled estrogen receptor-30 in human myometrial and uterine leiomyoma smooth muscle 
Ruijuan Tian, M.Sc., Zengyong Wang, M.Sc., Zhan Shi, M.D., Dong Li, Ph.D., Yuebing Wang, Ph.D., Yingjun Zhu, M.D., Ph.D., Wanjun Lin, M.D., Yu Gui, M.D., Ph.D., Xi-Long Zheng, M.D., Ph.D. 
Fertility and Sterility 
Volume 99, Issue 1, Pages e3 (January 2013)
DOI: /j.fertnstert
Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
G-protein-coupled estrogen receptor-30 (GPR30) is overexpressed in uterine leiomyomas. (A) Western blot detection of GPR30 expression using anti-GPR30 antibody without (left) and with (right) blocking peptide treatment. (B) Cumulative Western blot results showing GPR30 protein levels in human myometrium (HM) and leiomyoma (HL) tissues, standardized with α-tubulin. Insert: Representative Western blots (*P<.05, n = 9). (C) Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of GPR30 mRNA expression in myometrial and uterine leiomyoma tissues. The y-axis shows the expression level of GPR30 mRNA normalized with β-actin (*P<.05, n = 13).
Fertility and Sterility  , e3DOI: ( /j.fertnstert )
Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

3. Figure 2
Immunohistochemical detection of G-protein-coupled estrogen receptor-30 (GPR30) in uterine leiomyoma tissues. Tissue sections were stained with anti-GPR30 antibody and the secondary antibody conjugated with Alexa Fluor 568 (red) with the nuclei counterstained with DAPI (blue). Representative micrographs from tissue sections of uterine leiomyoma (HL) and matched myometrium (HM) were taken under a confocal microscope.
Fertility and Sterility  , e3DOI: ( /j.fertnstert )
Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
Estradiol (E2) increases G-protein-coupled estrogen receptor-30 (GPR30) mRNA in cultured smooth muscle cells (SMCs) derived from uterine leiomyoma. Primary cell culture of SMCs from myometrium and uterine leiomyoma was performed using the enzyme digestion approach. Protein and total RNA were extracted from cells from passages 3–5 for Western blot analysis and RT-qPCR, respectively. (A) Cumulative Western blot results showing the expression of GPR30 in cultured SMCs derived from human myometrium (HM) and uterine leiomyoma (HL) (*P<.01, n = 4). Insert: Representative Western blots showing the levels of GPR30 and α-tubulin. (B) Real-time quantitative polymerase chain reaction (RT-qPCR) data showing the mRNA levels of GPR30 in HL-SMCs and HM-SMCs (*P<.01, n = 4). (C) RT-qPCR data showing the mRNA levels of GPR30 in HL-SMCs and HM-SMCs in response to E2 at different concentrations as indicated on the x-axis for 24 hours. The y-axis represents the relative levels of GPR30 normalized by the HM-SMC control (*P<.05, n = 3). (D) RT-qPCR data showing the mRNA levels of GPR30 in HM-SMCs and HL-SMCs in response to treatment with E2 (100 nM) with and without ICI 182,780 (1 μM) as indicated (*P<.05, n = 3).
Fertility and Sterility  , e3DOI: ( /j.fertnstert )
Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

5. Figure 4
Activation of the mitogen-activated protein kinase (MAPK) pathway by G-protein-coupled estrogen receptor-30 (GPR30) agonist G-1. Cultured SMCs (passage 3–5) derived from human leiomyoma smooth muscle cells (HL-SMCs) and myometrium (HM-SMCs) were treated with and without G-1 (1 μM), E2 (100 nM), and PD98059 (10 μM) for 15 minutes as indicated, followed by extraction of protein for Western blot analysis. (A) Representative Western blots showing phosphorylation of p44/42 MAPK in HL-SMCs and HM-SMCs treated with or without G-1 and PD (B) Cumulative Western blot results showing phosphorylation of p44/42 MAPK in HL-SMCs and HM-SMCs with various treatments as indicated. The y-axis represents the relative levels of phospho-p44/42 MAPK normalized by total p44/42 MAPK (*P<.05, n = 4, vs. cells without any treatment). (C) Representative Western blots showing phosphorylation of p44/42 MAPK in HL-SMCs and HM-SMCs treated with or without E2 (100 nM). (D) Cumulative data showing the relative levels of phospho-p44/42 MAPK normalized by total p44/42 MAPK (*P<.05, n = 4, vs. control cells). (E) Representative Western blots showing phosphorylation of p44/42 MAPK in HL-SMCs and HM-SMCs treated with or without G-1 and E2. (F) Cumulative data showing the relative levels of phospho-p44/42 MAPK normalized by total p44/42 MAPK (*P<.05, n = 3).
Fertility and Sterility  , e3DOI: ( /j.fertnstert )
Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

6. Supplemental Figure 1
Estrogen receptor-α (ER-α) is highly expressed in uterine leiomyoma tissues. Human myometrial (HM) and uterine leiomyoma (HL) tissues were sampled from patients, and protein and RNA were extracted for Western blot and RT-qPCR, respectively. (A) Cumulative data showing the protein levels of ER-α normalized by α-tubulin in HM and HL tissues. Insert: Representative Western blots. (*P<.05, n = 9). (B) Real-time quantitative polymerase chain reaction (RT-qPCR) results showing the mRNA levels of ER-α in both tissues (*P<.05, n = 13).
Fertility and Sterility  , e3DOI: ( /j.fertnstert )
Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

7. Supplemental Figure 2
Characterization of primary cultured smooth muscle cells (SMCs) isolated from human leiomyoma (HL) and matched myometrial tissues (HM). SMC marker proteins, such as SM α-actin and calponin, were detected in cultured HL-SMCs and HM-SMCs using (A) immunostaining and (B) Western blot analysis. In immunostaining and fluorescent microcopy, the nuclei were counterstained with propidium iodide (PI).
Fertility and Sterility  , e3DOI: ( /j.fertnstert )
Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

8. Supplemental Figure 3
Immunocytochemical detection of G-protein-coupled estrogen receptor-30 (GPR30) in cultured smooth muscle cells (SMCs). The SMCs were cultured from human leiomyoma and its matched myometrium, fixed with 100% methanol and then stained with anti-GPR30 and the secondary antibody conjugated with Alexa Fluor 568 (red) with the nuclei counterstained with 4′,6-Diamidino-2-phenylindole (DAPI) (blue). Representative micrographs were acquired by confocal microscopy.
Fertility and Sterility  , e3DOI: ( /j.fertnstert )
Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions