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Jenny Seltmann, PhD, Lennart M

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1 IL-33 impacts on the skin barrier by downregulating the expression of filaggrin 
Jenny Seltmann, PhD, Lennart M. Roesner, PhD, Friedrich-Wilhelm von Hesler, MD, Miriam Wittmann, MD, Thomas Werfel, MD  Journal of Allergy and Clinical Immunology  Volume 135, Issue 6, Pages e4 (June 2015) DOI: /j.jaci Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 mRNA expression of FLG in monolayer keratinocytes and skin samples measured by quantitative RT-PCR. A, Ca2+-differentiated keratinocytes from healthy donors stimulated for 4 hours with 50 ng/mL of IL-4 (n = 7). B, Ca2+-differentiated and undifferentiated keratinocytes from healthy donors and patients with AD stimulated for 4 hours with 50 ng/mL of IL-33 as indicated (n = 3-7). C, Whole-skin samples stimulated with 50 ng/mL of IL-4 or IL-33 for 24 hours (n = 6). GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; ns, nonstimulated. Means and SEM are depicted. t test: *P < .05, **P < .005, ***P < .001. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Detection of FLG protein expression in the skin. Samples were stimulated with 50 ng/mL of IL-33 or IL-4 for 24 hours. A, Immunohistochemistry staining. Stained area within the epidermis except the stratum corneum (dotted line) was measured using cell Sens Dimension software (Olympus). B, Immunofluorescence staining. Fluorescence intensity within the epidermis except the stratum corneum (dotted line) was measured using ImageJ (NIH). n.s., Nonstimulated. Paired t test: *P < .05, **P < .005. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig E1 FLG expression in 3D skin equivalents. Skin equivalents were stimulated with IL-33 at the beginning of the air-liquid interphase for a total of 8 or 14 days. FLG staining in skin equivalents: 1 representative out of 7 independent experiments is shown. Time points of measurements as indicated. Stained area was quantified using cell Sens Dimension software (Olympus). ns, Not stimulated. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig E2 Penetration of Ara h 2 into the skin equivalent. Skin equivalents were either stimulated with IL-33 at the beginning of the air-liquid interphase for a total of 14 days or left unstimulated. Biotin-Ara h 2 that entered the skin equivalent was detected by streptavidin-PE. Area of positive staining is indicated by white arrows. auto, Autofluorescence; n.s., not stimulated; PE, phycoerythrin. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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