Iron overload–modulated nuclear factor kappa-B activation in human endometrial stromal cells as a mechanism postulated in endometriosis pathogenesis

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Presentation transcript:

Iron overload–modulated nuclear factor kappa-B activation in human endometrial stromal cells as a mechanism postulated in endometriosis pathogenesis 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 103, Issue 2, Pages 439-447 (February 2015) DOI: 10.1016/j.fertnstert.2014.10.046 Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 1 IκBα and p65 expression in response to iron overload in ESC. (A) Western blot picture of IκBα, p65, and β-actin proteins using ESC cytoplasmic extracts after 0.5 and 2 hours of exposure to iron overload. C = control condition. Protein weight is indicated in kilo-Daltons (kDa) (B) IκBα expression relative to β-actin bands. (C) p65 expression relative to β-actin bands. The bars show the mean ± SEM of five independent values (n = 5). Statistics are performed with one-way ANOVA with Dunnett's test (*P<.01 and **P<.05 vs. control condition). Fertility and Sterility 2015 103, 439-447DOI: (10.1016/j.fertnstert.2014.10.046) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 2 p65:DNA binding in response to iron overload in ESC. (A) Immunodetection assay for p65:DNA complexes (TransAM). The bars show the mean ± SEM of five independent values (n = 5) expressed as optical density measured at 450 nm. (B) The specificity of the assay was evaluated as described in Materials and Methods. J = Jurkat cells nuclear extracts; OligoNat = wild type κB oligonucleotide; OligoMut = mutated κB oligonucleotide.Statistics are performed with one-way ANOVA with Dunnett's test (*P<.05 vs. control condition). Fertility and Sterility 2015 103, 439-447DOI: (10.1016/j.fertnstert.2014.10.046) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 3 ICAM-1 expression in ESC and sICAM-1 levels in culture media of ESCs. (A) Western blot picture of ICAM-1 and β-actin using cytoplasmic extracts from ESCs. C = control condition. Protein weight is indicated in kilo-Daltons (kDa). (B) ICAM-1 expression relative to β-actin bands. (C) sICAM-1 ELISA using ESC conditioned medium. The bars show the mean concentration (pg/mg protein) ± SEM of five independent values (n = 5). Statistics are performed with one-way ANOVA with Dunnett's test (*P<.05 vs. control condition). Fertility and Sterility 2015 103, 439-447DOI: (10.1016/j.fertnstert.2014.10.046) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Effect of IKKβ inhibition before iron overload exposure in ESCs on IκBα and p65 cytoplasmic expression and p65:DNA binding. (A) Western blot picture of IκBα, p65, and β-actin proteins using ESC cytoplasmic extracts. (B) IκBα expression relative to β-actin bands. (C) p65 expression relative to β-actin bands. (D) Immunodetection assay for p65:DNA complexes (TransAM). The bars show the mean ± SEM of five independent values (n = 5) expressed as optical density measured at 450 nm. C = control condition (vehicle alone). Statistics are performed with one-way ANOVA followed by the Bonferroni post-test (*P<.05 and **P<.001 vs. vehicle; &P<.001, &&P<.05, and &&&P<.01 between conditions). Fertility and Sterility 2015 103, 439-447DOI: (10.1016/j.fertnstert.2014.10.046) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions