KRT76 interacts with Claudin1.

<p>(A) HIS-tagged KRT76 tail domain and HIS-tag alone where produced in E.coli, purified and immobilised on nickel-resin. Resin was then incubated with mouse paw pad lysates and the specific pull-down of CLDN1 with the KRT76- tail domain and not HIS-tag was shown by anti-Claudin1 WB. (B) Soluble extracts were prepared from A549 cells and anti-CLDN1 or non-immune IgG antibody immunoprecipitated. IP and lysate/input samples were then blotted for ZO-1, CLDN1 and KRT76. (C) A549 cells co-express CLDN1 and KRT76 and these colocalise in cytoplasmic punctate structures -see arrowheads.</p

<i>Krt76</i> gene trap disruption causes gross epidermal defects.

<p>(A) Schematic showing <i>Krt76</i> gene trap (knock-out first) targeting construct. (B) Whole mount LacZ staining of <i>Krt76^tm1a/+</i> reporter mice, shows <i>Krt76</i> expression in the dorsal and ventral snout and palate, eyelid, and vagina. (C) Mice homozygous for <i>Krt76</i> gene trap disruption (<i>Krt76^tm1a/tm1a</i>) exhibit flaky skin following birth (see arrow-insert). Adult <i>Krt76^tm1a/tm1a</i> mice exhibit a scruffy coat and smaller body weight (n = 3 males, age 9 weeks, ***p&lt;0.004) (D, E), as well as tail scaling (F). <i>Krt76^tm1a/tm1a</i> mice exhibit paw pad hyperpigmentation (G), concurring with regions of LacZ reporter expression (H). LacZ expression within paw pads is detected in exocrine glands (H′) and suprabasal epidermal layers (I). (J, J′) Haemotoxylin and Eosin (H&amp;E) staining of paw pads from WT (J) and <i>Krt76^tm1a/tm1a</i> (J′) mice. Yellow arrowheads indicate abnormal dermal pigmentation. (K, L) Immunofluorescence analysis with indicated antibodies in wild type and <i>Krt76^tm1a/tm1a</i> mouse paw pad. Samples are counter stained with nuclear dye DAPI (4',6-diamidino-2-phenylindole). Coloured brackets indicate approximate distribution of FLG and KRT76 expression around the granular layer. (M) Western blot analysis of WT and <i>Krt76^tm1a/tm1a</i> dorsal skin and face skin extracts. (N) Immunofluorescence analysis with anti-KRT76 and anti-K14 antibodies in wild type mouse dorsal skin at E14.5, E18.5, P1 and adult time points and adult <i>Krt76^tm1a/tm1a</i> dorsal skin (N′). Asterisks indicate non-specific basal layer staining. (O) <i>Krt76</i> mRNA qRT-PCR analysis of p3 dorsal skin relative to <i>Gapdh</i>. Scale bars represent 50 µm.</p

<i>Krt76^mutant</i> mice show barrier function defects and KRT76 stabilises Claudin1 at tight junctions.

<p>(A) Transepidermal water loss assay on P3 dorsal skin from wild type and <i>Krt76^tm1a/tm1a</i> mice. (B) P3 paw pad skin was dermally injected with a biotin tracer and diffusion through the epidermis assessed, with Filaggrin (FLG) and DAPI co-staining for tissue orientation. Yellow arrowhead shows diffusion in suprabasal keratinocytes into cornified layer. (C) Biotin tracer was assessed alongside TJ component, Claudin1 (CLDN1). Tracer exclusion indicated by flanking yellow arrowheads. (D) Immunofluorescence analysis of CLDN1 and Ecadherin (ECAD) distribution in wild type and <i>Krt76^tm1a/tm1a</i> mouse dorsal skin. (E) Image quantification at the cellular surface shows an inward shift and a decrease in intensity of CLDN1 not observed with ECAD. (F) Further quantification by image analysis of CLDN1 co-localisation at the cell surface with ECAD or DAPI in the nucleus. (G, H) Immunofluorescence analysis of CLDN1 localization in dorsal skin of wild-type and <i>Krt76^tm1a/tm1a</i> mice in early phenotype and biopsy wounded adult dorsal skin of wild-type and <i>Krt76^tm1a/tm1a</i> mice. (I) Dorsal skin fractionation assay showing localisation of different proteins to different fraction; relative lcoalisation of CLDN1 are indicated in (I′). (J, K) Immunofluorescence analysis of CLDN1 localization in adult dorsal skin and paw pads of 4OHT-treated conditional <i>Krt76</i> knock-out mice and control sibling. Note paw pad phenotype from grooming transfer of tamoxifen. *p&lt;0.05, **p&lt;0.01. Error bars  =  S.E.M. Scale bars represent 50 µm.</p

Additional file 3: Figure S3. of The polarity protein Scrib mediates epidermal development and exerts a tumor suppressive function during skin carcinogenesis

Analysis of Scrib-deficient DMBA/TPA-driven epidermal lesions. (A) Kaplan Meier survival plot for DMBA/TPA treated Scrib +/+ , Scrib +/fl and Scrib fl/fl mice. The average survival of Scrib fl/fl mice (23.5 weeks post-DMBA) was comparable to Scrib +/+ and Scrib +/fl mice (24.5 and 24.9 weeks post-DMBA respectively). No statistical difference in survival was observed between genotypes (χ2 = 0.029 – 2.15, df = 1, P ≥ 0.1426, Log-rank Mantel-Cox test, n = 12–16). (B) qRT-PCR for Scrib mRNA confirmed a significant reduction in Scrib +/fl and Scrib fl/fl volume-matched (40 – 80 mm3) early benign papillomas compared to Scrib +/+ lesions (*p < 0.0001, unpaired t-test, n = 3). (C) IF to detect Scrib (green) and DAPI (blue) in Scrib +/+ , Scrib +/fl and Scrib fl/fl DMBA/TPA-induced size-matched benign papillomas (n = 3, scale bar = 50 μm, insert 1–3 scale bar = 10 μm). Representative p-ERK IHC images (D) and quantitation (E) from Scrib +/+ , Scrib +/fl and Scrib fl/fl DMBA/TPA-induced size-matched benign papillomas (scale bar = 50 μm, P ≥ 0.5919, unpaired t-test, error bars = SD, n = 3). (F) IF to detect ZO-1 (green), E-cadherin (red) and DAPI (blue) in Scrib +/+ , Scrib +/fl and Scrib fl/fl DMBA/TPA-induced size-matched benign papillomas (n = 3, scale bar = 50 μm). (PPTX 1616 kb

Additional file 1: Figure S1. of The polarity protein Scrib mediates epidermal development and exerts a tumor suppressive function during skin carcinogenesis

Cell polarity is not deregulated in Scrib KO embryonic epidermis. (A) IF to detect pan-Dlg (green) and DAPI (blue) and (B) IF to detect E-cadherin (red) and DAPI (blue) in Scrib Wt, Het and KO embryonic epidermis at E17.5 (n = 3, scale bar = 50 μm, dashed line represents basement membrane). (PPTX 539 kb