Endometrial expression and in vitro modulation of the iron transporter divalent metal transporter-1: implications for endometriosis Carlos Patricio Alvarado-Díaz,

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Endometrial expression and in vitro modulation of the iron transporter divalent metal transporter-1: implications for endometriosis Carlos Patricio Alvarado-Díaz, Ph.D., Marco Tulio Núñez, Ph.D., Luigi Devoto, M.D., Reinaldo González-Ramos, M.D., Ph.D. Fertility and Sterility Volume 106, Issue 2, Pages 393-401 (August 2016) DOI: 10.1016/j.fertnstert.2016.04.002 Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Immunohistochemical detection of DMT1 across the menstrual cycle in endometrial sections of tissue from healthy women (H) and endometriosis patients (E). Signal from DMT1 detection (reddish brown) is observed in glandular (yellow arrows) and stromal (red asterics) compartments. Sections incubated with IgG are shown as negative control (C-). Scale bar = 100 μm. Fertility and Sterility 2016 106, 393-401DOI: (10.1016/j.fertnstert.2016.04.002) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Endometrial DMT1 protein expression across the menstrual cycle in healthy women and endometriosis patients. (A) Western blot of DMT1 using protein extracts from endometrial samples at distinct menstrual phases. Beta-actin was used as a control of loaded protein. In the image, three representative samples from each analyzed group (n = 8) are shown. Molecular weight of revealed bands (kDa) is shown. (B) Densitometric analysis of DMT-80, DMT-65, DMT-55, and DMT-50 bands with respect to the corresponding β-actin band. AU = Arbitrary units. Boxes show median, 25th and 75th percentile, and range for eight samples from healthy women (H, yellow bars) and endometriosis patients (E, red bars) in each menstrual phase. *P<.05, **P<.01, and ***P<.001 for comparisons of H vs. E in each phase; &P<.05 and &&&P<.001 for comparisons between different phases in healthy or endometriosis tissue samples. Fertility and Sterility 2016 106, 393-401DOI: (10.1016/j.fertnstert.2016.04.002) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Effect of a 24-hour iron overload stimulus over DMT1 and Fn-L expression. (A) Representative images of WB using ESC cytoplasmic extracts. Beta-actin was used as a control of loaded protein. C = control condition, +Fe = FeSO4 50 μM. Molecular weight of revealed bands are shown. (B) Densitometric analysis of bands in (A) normalized to the corresponding β-actin band. The bars show the mean ± SEM of five independent values (n = 5). *P<.05 vs. the control condition. Fertility and Sterility 2016 106, 393-401DOI: (10.1016/j.fertnstert.2016.04.002) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Effect of a 24-hour IL-1β stimulus over DMT1 and Fn-L expression. (A) Representative image of WB using ESC cytoplasmic extracts. Beta-actin was used as a control of loaded protein. C = control condition, +IL-1β = IL-1β 45 pg/mL. Molecular weights (kDa) of revealed bands are shown. (B) Densitometric analysis of bands in (A) normalized to the corresponding β-actin band. The bars show the mean ± SEM of five independent values (n = 5). *P<.05 vs. the control condition. Fertility and Sterility 2016 106, 393-401DOI: (10.1016/j.fertnstert.2016.04.002) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions