1. Altered E-Cadherin Levels and Distribution in Melanocytes Precede Clinical Manifestations of Vitiligo 
Roselyne Y. Wagner, Flavie Luciani, Muriel Cario-André, Alain Rubod, Valérie Petit, Laila Benzekri, Khaled Ezzedine, Sébastien Lepreux, Eirikur Steingrimsson, A. Taieb, Yvon Gauthier, Lionel Larue, Véronique Delmas 
Journal of Investigative Dermatology 
Volume 135, Issue 7, Pages (July 2015)
DOI: /jid
Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
E-cadherin (Ecad) staining is altered in vitiligo melanocytes. (a) Staining of Ecad (green) and Trp2 (red, specific of melanocytes) of the epidermis. A simplified sketch shows the merge. Type 1: Ecad staining is homogeneously distributed in melanocyte; type 2: Ecad staining is heterogeneous; and type 3: Ecad staining is absent. Scale bars=20 μm. The dotted lines represent the basal membrane. Bars=20 μm. (b, c) Mean percentages of control (blue) and vitiligo patient (red) melanocytes displaying type 1, 2, and 3, and types 2 and 3 (2–3) labeled with Ecad and β-catenin (bcat)-specific antibodies. (d) Staining of Ecad (green) and Melan A (red) of reconstructed epidermis made with control keratinocytes and either control (RE mel crtl) or nonlesional vitiligo (RE mel vit) melanocytes. Bars=20 μm. (e) Mean percentages of control (blue) and vitiligo (red) melanocytes displaying Ecad type 1 or type 2–3. **P<0.01, ***P<0.001, ****P<
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
The amount of E-cadherin (Ecad) correlates with the numbers of melanocytes of the upper layer of the epidermis. (a) Human epidermis from controls (Ctrl) and vitiligo patients (Vit) labeled with anti-Ecad and Trp2 antibodies and DAPI (4',6-diamidino-2-phenylindole; blue). Basal melanocytes are indicated by white arrowheads and suprabasal melanocytes by white arrows. Bars=20 μm. (b) Numbers of melanocytes (Mc) in control (blue) and nonlesional vitiligo skin sections (red). (c) Inverse correlation between the number of suprabasal melanocytes and Ecad; Pearson's correlation coefficient of Red dots indicate vitiligo patients and blue dots indicate controls. (d) Number of keratinocytes between two melanocytes in the epidermis of controls and in nonlesional skin of vitiligo patients. Each individual score is represented as a dot (blue=control, red=vitiligo). On the right, the number of scores of each value is indicated. Means of the number of keratinocytes are represented by horizontal bars: 6±0.3 for controls and 9±0.4 for vitiligo. **P<0.01, ****P<0.0001; NS, nonsignificant.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Loss of E-cadherin (Ecad) favors murine melanocyte detachment after skin friction. (a) Hair and tail pigmentation in wild-type (WT) mice and mice lacking Ecad in melanocytes (ΔEcad). Dramatic depigmentation zone (arrow). (b) Immunostaining of tail skin of controls and ΔEcad mice in a Dct::LacZ genetic background using anti-Ecad (green) and β-galactosidase (βgal; red) antibodies. Scale bar=10 μm. Basement membrane is indicated as a dotted line. Bars=20 μm. (c, d) Transmission electron micrographs of WT and ΔEcad epidermal mouse melanocytes. Bars=2 μm (c) and human control and vitiligo epidermal melanocytes from nonlesional skin (d). Note the large vacuoles, black arrows, in the cytoplasm of murine ΔEcad and human pre-vitiligo melanocytes. (e) Photographs of WT and ΔEcad tails before (d0) and after repeated friction (d200). Tails were brushed 5 days a week for 28 weeks. Boundary between the brushed (B) and nonbrushed (NB) parts of the tail is indicated. (f) Numbers of melanocytes found in NB and B parts for each genotype. Tail sections 866, 980, 624, and 550 corresponding to WT-B, WT-NB, ΔEcad-B, and ΔEcad-NB, respectively, were generated from three WT and three ΔEcad mice. Histograms represent the ratio between the number of melanocytes found in NB and B parts and that in the NB part for each genotype, as percentages. (g) Location of melanocytes within the epidermis of the B and NB epidermis. Note that some melanocytes were found in the suprabasal epidermis after friction. Bars=20 μm. (h) Percentage of detached melanocytes (Mc). The number of sections per mouse tail and the number of mice are given in f. **P<0.01, ***P<0.001, ****P<0.0001; NS, nonsignificant.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Loss of E-cadherin (Ecad) affects melanoblast proliferation during embryogenesis specifically in the epidermis. (a) Transverse sections of wild-type (WT) and ΔEcad tails of p10 (postnatal day 10) mice in a Dct::LacZ genetic background. Melanocytes are identified as β-galactosidase-positive cells. Bars=50 μm. (b) Scatter dot plot reporting the number of melanocytes (Mc) in sections of WT and ΔEcad tails at p10. Each dot represents the average of four sections from the same mouse. Five mice of each group were studied. (c) Number of melanoblasts in the epidermis and the dermis of WT and ΔEcad mouse tails at E15.5. The number of melanoblasts in the epidermis and dermis found in mouse tails at E15.5 was determined for four WT and three ΔEcad embryos. The numbers of melanoblasts in the epidermis, but not in the dermis, were smaller in ΔEcad compared with WT embryos. (d) Proliferation rate of Dct::lacZ-positive cells at E15.5 in the tail, as evaluated by measuring BrdU incorporation as previously described (Luciani et al., 2011). Approximately 70 sections, derived from two to four embryos from independent litters, were analyzed for each genotype. No apoptotic melanoblasts were detected at E15.5 in either mutants or controls (data not shown). *P<0.05, ****P<0.0001; NS, nonsignificant.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Loss of E-cadherin (Ecad) from the melanocyte membrane and melanocyte detachment in reconstructed human epidermis after exposure to hydrogen peroxide (H202). (a, b) Immunostaining of control (a) and vitiligo (b) biopsies using anti-4-HNE (red) and Trp2 (green) antibodies. DAPI (4',6-diamidino-2-phenylindole; blue). Bars=20 μm. (c–h) Human MNT-1 melanoma cells incubated with (f–h) or without (c–e) 0.025% H202 for 6 hours and labeled with anti-Ecad (green), Melan A (red) antibodies, and DAPI (blue). (c–h) Ecad, (d, g) Ecad+DAPI, (e, h) Ecad+Melan A. Bars=10 μm. (i, j) Percentages of basal (i) and detached (j) melanocytes (Mc) in reconstructed epidermis with normal Sh Ecad (ShEcad, red) or Puro (Ctrl, blue) transduced melanocytes. Reconstructed epidermis was treated or not with 0.025% H202 for 6 hours. (k) Reconstructed epidermis with ShEcad transduced or Ctrl melanocytes incubated with or without 0.025% H202 for 6 hours and labeled with anti-Ecad (green) and melan A antibodies (red). Ecad labeling is weak at the melanocyte membrane in ShEcad samples without H202 (arrows) and in Ctrl melanocytes after H202 treatment. Ecad labeling was totally absent from ShEcad melanocyte-exposed H202 (star). The dotted line indicates the epidermis–dermis junction. Bars=20 μm. *P<0.05; NS, nonsignificant.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions