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Cadherin 11, a miR-675 Target, Induces N-Cadherin Expression and Epithelial– Mesenchymal Transition in Melasma Nan-Hyung Kim, Soo-Hyun Choi, Tae Ryong Lee, Chang-Hoon Lee, Ai-Young Lee Journal of Investigative Dermatology Volume 134, Issue 12, Pages (December 2014) DOI: /jid Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions
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Figure 1 MiR-675 inhibits CDH11 expression by targeting its 3′-untranslated region (3′-UTR). (a) The complementary sequences between miR-675 and the 3′-UTR of CDH11 messenger RNA. The relative ratios of luciferase activities in cultured normal human fibroblasts, which were transfected with pMIR-CDH11 or mutants with or without miR-675. *P<0.05. Data represent the means±SD of five independent experiments. (b, c) Western blot analysis of CDH11 expression in fibroblasts and keratinocytes without treatment (b) and treated with different concentrations of the miR-675 mimic or inhibitor (c). β-Actin was used as an internal standard. (d) Real-time PCR results for the relative ratios of CDH11 expression levels in hyperpigmented compared with normally pigmented skin from five melasma patients with miR-675 reduction. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal standard. (e) Representative immunofluorescent staining of hyperpigmented and normally pigmented skin from four melasma patients with miR-675 reduction using anti-CDH11 and anti-K14 antibodies (bar=0.1mm). Journal of Investigative Dermatology , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions
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Figure 2 CDH11 expression in fibroblasts is involved in melanogenesis. (a) A schematic of fibroblast–melanocyte (FB–MC) cocultures, representing a monolayer coculture, a coculture with fibroblasts in the lower chamber and melanocytes in the upper insert, and a coculture with fibroblasts on the outer side of the insert and melanocytes on the inner side of the insert. (b) Western blot analysis of CDH11 expression after the transfection of cells with the CDH11 gene or CDH11 small interfering RNA (siRNA). (c) Western blot analysis of tyrosinase, MITF, CREB, p-CREB, ERK, p-ERK, AKT, p-AKT, Wnt-3A, and β-catenin expression (with or without LY treatment) in melanocytes cocultured with CDH11-overexpressing or CDH11 knockdown fibroblasts. (d) Western blot analysis of β-catenin expression in membranous and nuclear fractions of monolayer cocultures with CDH11-overexpressing or CDH11 knockdown fibroblasts. (e) Immunoprecipitation using an anti-CDH11 antibody in monolayer cocultures with CDH11 knockdown fibroblasts. (f) Immunofluorescence staining of monolayer cocultures with CDH11 knockdown fibroblasts using anti-CDH11 and anti-β-catenin antibodies (bar=0.1mm). *P<0.05. Data represent the means±SD of five independent experiments. Journal of Investigative Dermatology , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions
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Figure 3 CDH11 expression in keratinocytes is involved in melanogenesis. (a) Western blot analysis of CDH11 expression after transfection of cells with the CDH11 gene or CDH11 small interfering RNA (siRNA). (b, c) Western blot analysis of tyrosinase, MITF, cAMP-responsive–element-binding protein (CREB), p-CREB, extracellular signal–regulated kinase (ERK), p-ERK, AKT, p-AKT, Wnt-3A, and β-catenin expression (with or without LY treatment) in melanocytes cocultured with CDH11-overexpressing or CDH11 knockdown keratinocytes. (d) Western blot analysis of β-catenin expression in the cytoplasmic and nuclear fractions of melanocytes cocultured with CDH11-overexpressing keratinocytes. (e) Immunoprecipitation using an anti-CDH11 antibody in cocultures with CDH11 knockdown keratinocytes. *P<0.05. Data represent the means±SD of five independent experiments. Journal of Investigative Dermatology , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions
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Figure 4 CDH11 in fibroblasts induces N-cadherin expression in melanocytes. (a, b) Western blot analysis of N-cadherin and E-cadherin expression in different settings of CDH11-overexpressing (a) or CDH11 knockdown (b) fibroblast–melanocyte (FB–MC) cocultures. (c, d) Immunofluorescent staining of monolayer cocultures with CDH11-overexpressing (c) or CDH11 knockdown (d) fibroblasts using anti-CDH11 and anti-N-cadherin antibodies (bar=0.1mm). (e) Immunoprecipitation using anti-CDH11 antibody in monolayer cocultures with CDH11 knockdown fibroblasts. (f) Western blot analysis of Twist1 expression in CDH11-overexpressing fibroblasts in the absence or presence of melanocytes. (g) Western blot analysis of tyrosinase expression from melanocytes cocultured with CDH11-overexpressed fibroblasts with or without N-cadherin knockdown. (h) Boyden chamber assay for melanocyte migration in the presence of CDH11-overexpressing or CDH11 knockdown fibroblasts. *P<0.05. Data represent the means±SD of five independent experiments. siRNA, small interfering RNA. Journal of Investigative Dermatology , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions
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Figure 5 CDH11 in keratinocytes induces epithelial–mesenchymal transition. Western blot analysis of N-cadherin, E-cadherin, and Twist1 expression in CDH11-overexpressing keratinocytes (KC) with or without melanocytes (MC). *P<0.05. Data represent the means±SD of five independent experiments. Journal of Investigative Dermatology , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions
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