Anca Chelariu-Raicu, M. D. , Cornelia Wilke, Ph. D. , Melanie Brand, M

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Syndecan-4 expression is upregulated in endometriosis and contributes to an invasive phenotype Anca Chelariu-Raicu, M.D., Cornelia Wilke, Ph.D., Melanie Brand, M.Sc., Anna Starzinski-Powitz, Ph.D., Ludwig Kiesel, M.D., Ph.D., Andreas N. Schüring, M.D., Martin Götte, Ph.D. Fertility and Sterility Volume 106, Issue 2, Pages 378-385 (August 2016) DOI: 10.1016/j.fertnstert.2016.03.032 Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Representative pictures of immunohistochemical stainings for syndecan-4 protein expression in endometrium (original magnification, ×320). (A) Negative antibody control using goat IgG. (B) Syndecan-4 staining in proliferative phase endometrium (nonendometriotic control group). (C) Syndecan-4 staining in secretory phase endometrium (nonendometriotic control group). (D) Epithelial cell layer stained positive for syndecan-4 in eutopic endometrium of a patient with endometriosis. (E) High epithelial and stromal expression of syndecan-4 in eutopic endometrium of a patient with endometriosis. (F) Strong staining of an atypical endometriotic epithelial cell in eutopic endometrium of a patient with endometriosis (arrow). Insert: Syndecan-4-positive single cells in eutopic endometrium of a patient with endometriosis. Fertility and Sterility 2016 106, 378-385DOI: (10.1016/j.fertnstert.2016.03.032) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Boxplot of semiquantitative analysis of immunohistochemistry scoring of syndecan-4 staining. (A) Analysis of tubular glands. (B) Analysis of surrounding dense stroma. Endometriosis group: n = 44. Control group: n = 62; *P<.001. Fertility and Sterility 2016 106, 378-385DOI: (10.1016/j.fertnstert.2016.03.032) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Syndecan-4 is a regulator of invasive growth. The endometriotic cell line 12Z was transfected with a negative control siRNA or siRNA against syndecan-4 (SDC-4) and analyzed for changes in cell behavior and invasion-related gene expression. (A) qPCR analysis reveals a significant downregulation of RAC1 and ATF2 expression in syndecan-4-depleted 12Z cells. Data are normalized to 18S rRNA expression and shown as fold change relative to control siRNA transfected cells (n ≥ 3; error bars = SEM; **P<.01; *P<.05). (B) Syndecan-4 depletion does not affect cell viability as determined by MTT assay (n = 3; error bars = SD; P = not significant). (C) Syndecan-4 siRNA-depletion reduces invasiveness of 12Z cells in the matrigel chamber assay (n = 10; error bars = SEM; *P<.05). (D) Representative stained matrigel invasion assay filters demonstrating reduced invasiveness of syndecan-4-depleted 12Z cells. (E) Syndecan-4 siRNA knockdown induces a downregulation of MMP3 expression in 12Z cells, as determined by qPCR (n = 3; error bars = SEM; *P<.05). Fertility and Sterility 2016 106, 378-385DOI: (10.1016/j.fertnstert.2016.03.032) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions