Epidermal growth factor–containing fibulin-like extracellular matrix protein 1 expression and regulation in uterine leiomyoma Erica E. Marsh, M.D., M.S.C.I.,

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Epidermal growth factor–containing fibulin-like extracellular matrix protein 1 expression and regulation in uterine leiomyoma Erica E. Marsh, M.D., M.S.C.I., Shani Chibber, M.S., Ju Wu, M.D., Kendra Siegersma, B.S., Julie Kim, Ph.D., Serdar Bulun, M.D. Fertility and Sterility Volume 105, Issue 4, Pages 1070-1075 (April 2016) DOI: 10.1016/j.fertnstert.2015.12.004 Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 1 In vivo expression of epidermal growth factor–containing fibulin-like extracellular matrix protein 1 (EFEMP1) and fibulin-3. (A) Real-time polymerase chain reaction (PCR) of EFEMP1 gene expression in myometrial tissue (MYO) versus matched leiomyoma tissue (LEIO), showing decreased expression in LEIO. (B) Immunohistochemical staining of MYO and LEIO with fibulin-3 antibody versus control, demonstrating decreased staining in LEIO than in MYO. The control slides were stained with antimouse secondary antibody with no primary antibody. Stains are provided at ×10 and ×40 (insets) magnification. Fertility and Sterility 2016 105, 1070-1075DOI: (10.1016/j.fertnstert.2015.12.004) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 2 In vitro expression of EFEMP1 and fibulin-3. (A) Real-time PCR of EFEMP1 gene expression in human MYO versus matched LEIO showing decreased expression in LEIO. (B) Immunoblot of human MYO and LEIO, demonstrating decreased fibulin-3 protein in LEIO than in MYO. Abbreviations as in Figure 1. Fertility and Sterility 2016 105, 1070-1075DOI: (10.1016/j.fertnstert.2015.12.004) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 3 In vitro immunofluoroscopy of fibulin-3. Markedly decreased expression of fibulin-3 is seen in LEIO compared with matched MYO. Panels A and E demonstrate staining of DAPI. B and F demonstrate α-smooth muscle actin stains for cytoplasm, because EFEMP1 is largely located in the cytoplasm. C and G demonstrate fibulin-3 staining for levels of MYO staining compared with LYO staining. D and H demonstrate a merged image of all stains. Abbreviations as in Figure 1. Fertility and Sterility 2016 105, 1070-1075DOI: (10.1016/j.fertnstert.2015.12.004) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Primary LEIO were treated with varying doses of 5-aza-2′-deoxycytidine (AZA) for 5 days, and real-time PCR was performed to determine impact on EFEMP1 expression. *P<.001 compared with vehicle. Abbreviations as in Figure 1. Fertility and Sterility 2016 105, 1070-1075DOI: (10.1016/j.fertnstert.2015.12.004) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions