Knockdown of Filaggrin Impairs Diffusion Barrier Function and Increases UV Sensitivity in a Human Skin Model Michael Mildner, Jiang Jin, Leopold Eckhart,

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Knockdown of Filaggrin Impairs Diffusion Barrier Function and Increases UV Sensitivity in a Human Skin Model Michael Mildner, Jiang Jin, Leopold Eckhart, Sanja Kezic, Florian Gruber, Caterina Barresi, Caroline Stremnitzer, Maria Buchberger, Veronika Mlitz, Claudia Ballaun, Barbara Sterniczky, Dagmar Födinger, Erwin Tschachler Journal of Investigative Dermatology Volume 130, Issue 9, Pages 2286-2294 (September 2010) DOI: 10.1038/jid.2010.115 Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Expression of filaggrin is efficiently blocked by small interfering RNA (siRNA) transfection in organotypic skin cultures. After 7 days in culture, organotypic skin was analyzed by reverse transcription-PCR, western blot analysis, and immunofluorescence staining. (a) Filaggrin mRNA production in filaggrin-deficient organotypic skin culture was reduced by more than 60% as compared with control samples. Error bars represent one SD calculated from three replicates for each set of values (*P<0.001). (b) In contrast to untreated, mock-treated, or scrambled siRNA-treated organotypic skin cultures, filaggrin protein expression was almost completely abolished in organotypic skin cultures transfected with filaggrin-specific siRNAs. Equal loading of protein was controlled by the analysis of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. (c) Immunofluorescence staining revealed pronounced filaggrin staining in the granular layers of control organotypic skin cultures, whereas filaggrin expression was virtually absent in organotypic skin cultures from filaggrin siRNA-transfected keratinocytes. One representative experiment of three is shown. Bars=40μm. Journal of Investigative Dermatology 2010 130, 2286-2294DOI: (10.1038/jid.2010.115) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Filaggrin deficiency leads to the loss of keratohyalin granules and impaired lamellar body formation. (a, b) Hematoxylin and eosin staining of organotypic skin cultures that were untreated, mock treated, or transfected with scrambled small interfering RNA (siRNA), as well as organotypic skin cultures deficient for filaggrin, is shown. Compared to the controls (left panel), keratohyalin granules are completely missing in the filaggrin-deficient organotypic skin cultures (right panel). One representative experiment of three is shown. Bars=100μm (a), 40μm (b). Transmission electron microscopy reveals fewer and smaller keratohyalin granules ((c) arrows) and disturbed lamellar body (LB) formation in the filaggrin-deficient organotypic skin cultures (d). Note, most lamellar bodies are smaller and typical lamellae are not visible (d). Des, desmosome. Bars=2μm (c), 100nm (d). (e) Number of keratohyaline granules per cell was counted. (f) Area of lamellar bodies was measured. Journal of Investigative Dermatology 2010 130, 2286-2294DOI: (10.1038/jid.2010.115) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Filaggrin knockdown in organotypic skin cultures does not affect keratinocyte differentiation or the lipid composition. (a) Expression of differentiation-associated proteins was analyzed by western blotting. Except of an increased SPRR2A expression all other proteins were not regulated by filaggrin knockdown. Equal loading of protein was controlled by Ponceau staining of the membrane (data not shown), and western blot analysis of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). One representative experiment of three is shown. (b) Total lipid composition was analyzed by thin layer chromatography. The following solvent systems were used sequentially: upper panel: chloroform/methanol/water 40:10:1 (v/v/v) to 10cm, chloroform/methanol/acetic acid 190:9:1 (v/v/v) to 16cm, and hexane/diethylether/acetic acid 70:30:1 (v/v/v) to 20cm. Middle panel: chloroform/acetone/methanol 76:4:20 (v/v/v) to 2cm, chloroform/acetone/methanol 80:10:10 (v/v/v) to 15cm, and chloroform/diethylether/ethylacetate/methanol 72:6:20:2 (v/v/v) to 18cm. Lower panel: hexane/toluene 1:1 (v/v) to 6cm and hexane/diethyl ether/acetic acid 80:20:1 (v/v) to 9cm. Journal of Investigative Dermatology 2010 130, 2286-2294DOI: (10.1038/jid.2010.115) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Keratin solubility is not affected by filaggrin knockdown. The solubility of keratins 1, 2, and 10 was analyzed by fractionated lysing of the organotypic skin cultures and western blot analysis. Equal amounts of scrambled small interfering RNA (siRNA) control or filaggrin knockdown organotypic skin cultures were used for the solubility study. Pellets from NP40 buffer lysates were extracted with urea. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) western blot analysis of the corresponding NP40 supernatants confirmed the comparability of the aliquots (data not shown). (a) Similar amounts of keratins 1 and 10 were detectable in each urea fraction in both control and siRNA-treated samples. (b) The remaining insoluble keratins after 8M urea extraction were further completely resolved. High amounts of keratins 1, 2, and 10 were detected in both filaggrin knockdown organotypic skin cultures and control samples. One representative experiment of three is shown. Journal of Investigative Dermatology 2010 130, 2286-2294DOI: (10.1038/jid.2010.115) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 The epidermal diffusion barrier function is disturbed in filaggrin-deficient organotypic skin cultures. After 7 days in culture, 20μl of the fluorescent dye Lucifer yellow was applied onto the stratum corneum of the organotypic skin cultures and the penetration of the green dye was investigated in thin sections under the fluorescence microscope. Lucifer yellow penetrated through the stratum corneum down to the basal layer of the filaggrin-deficient epidermis but was retained in the stratum corneum of the control samples. Bars=100μm. Journal of Investigative Dermatology 2010 130, 2286-2294DOI: (10.1038/jid.2010.115) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Decreased urocanic acid (UCA) production and increased UV sensitivity in filaggrin-deficient organotypic skin cultures. (a) After 7 days in culture, organotypic skin were lysed in 1M KOH and subjected to HPLC. Filaggrin deficiency resulted in a 60% reduction of the UCA content in the filaggrin knockdown samples. Error bars represent one SD calculated from three experimental replicates for each set of values each carried out in duplicate (*P<0.001). (b) Exposure to UVB (20mJcm−2) led to an increased cyclobutane pyrimidine dimer (CPD) formation in filaggrin knockdown samples after exposure to UVB. Error bars represent one SD calculated from two experimental replicates for each set of values each carried out in triplicate (*P<0.01). (c) Immunohistochemistry for active caspase-3 revealed an enhanced activation of caspase-3, a marker of apoptosis, in filaggrin-deficient organotypic skin cultures after exposure to UVB (100 and 150mJcm−2). Bars=100μm. Journal of Investigative Dermatology 2010 130, 2286-2294DOI: (10.1038/jid.2010.115) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions