Development of an Ichthyosiform Phenotype in Alox12b-Deficient Mouse Skin Transplants Silvia de Juanes, Nikolas Epp, Susanne Latzko, Mareen Neumann,

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Development of an Ichthyosiform Phenotype in Alox12b-Deficient Mouse Skin Transplants Silvia de Juanes, Nikolas Epp, Susanne Latzko, Mareen Neumann, Gerhard Fürstenberger, Ingrid Hausser, Hans-Jürgen Stark, Peter Krieg Journal of Investigative Dermatology Volume 129, Issue 6, Pages 1429-1436 (June 2009) DOI: 10.1038/jid.2008.410 Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Ichthyosiform phenotype of mature Alox12b-deficient mouse skin. (a, b) Gross morphology shows no differences between control and 12R-LOX−/− mouse skin grafted on nude mice for 4 weeks. (c, d) Representative skin sections from control and mutant mouse skin grafts stained with hematoxylin and eosin. The control skin graft is almost identical to normal adult mouse skin. In contrast, the grafted 12R-LOX−/− mouse skin shows epidermal acanthosis and severe hyperkeratosis. Scale bars=0.1mm. Journal of Investigative Dermatology 2009 129, 1429-1436DOI: (10.1038/jid.2008.410) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Ultrastructural anomalies in Alox12b deficient skin transplants. Ultrastructure of the grafted skin from control (a, c) and 12R-LOX−/− (b, d, e) neonates. (b) Stratum corneum and stratum granulosum of the grafted mutant skin. Note the markedly piled up cornified cells and the increased size and number of keratohyalin granules. (d) Lipid droplets (arrows) and nuclear remnants (asterisk) in the stratum corneum. (e) Remnants of cytoplasmatic organelles in the intercellular space of the stratum corneum. Scale bars (a, b)=10μm, (c)=1μm, (d)=2μm, (e)=50nm. Journal of Investigative Dermatology 2009 129, 1429-1436DOI: (10.1038/jid.2008.410) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Hyperproliferative phenotype of mature Alox12b-deficient mouse skin. Immunofluorescence on (a, c) control and (b, d) 12R-LOX−/− skin grafts shows an increased number of Ki67-positive nuclei (b) and strong upregulation of keratin K6 throughout the epidermis (d) in mutant grafts. Nuclei are counterstained with Hoechst 33258. The dashed line shows the epidermal/dermal junction. Scale bars (a, b)=20μm, (c, d)=25μm. Journal of Investigative Dermatology 2009 129, 1429-1436DOI: (10.1038/jid.2008.410) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Analysis of epidermal differentiation markers in transplanted skin from control and Alox12b-deficient mice. Grafted skin sections from control (left panel) and Alox12b-deficient mice (right panel) were immunostained for the indicated epidermal differentiation markers. Nuclei are counterstained with Hoechst 33258. (a, b) Immunostaining of keratin 14 is seen in similar levels at the basal layer. (c, d) Keratin 10, (e, f) filaggrin, (g, h) repetin, and (i, j) involucrin staining are found in their typical localizations in control as well as in mutant skin grafts but appeared broader in the transplants from deficient skin. Scale bars (a–j)=20μm. Journal of Investigative Dermatology 2009 129, 1429-1436DOI: (10.1038/jid.2008.410) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Unimpaired filaggrin processing and upregulation of CE proteins in mature Alox12b-deficient mouse skin. Equal amounts of epidermal protein extracts from control (+/+) and homozygous mutant (-/-) skin graft were separated through SDS–PAGE and subjected to western blot analysis using specific antibodies against (a) involucrin (Inv), repetin (Rptn), keratin 6 (K6), keratin 10 (K10), keratin 5 (K5), glycerol-aldehyd-3-phosphat-dehydrogenase (GAPDH), and (b) filaggrin (Fil). Journal of Investigative Dermatology 2009 129, 1429-1436DOI: (10.1038/jid.2008.410) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions