1. Elevated ΔNp63α Levels Facilitate Epidermal and Biliary Oncogenic Transformation 
Michael Devos, Barbara Gilbert, Geertrui Denecker, Kirsten Leurs, Conor Mc Guire, Kelly Lemeire, Tino Hochepied, Marnik Vuylsteke, Jo Lambert, Caroline Van Den Broecke, Louis Libbrecht, Jody Haigh, Geert Berx, Saskia Lippens, Peter Vandenabeele, Wim Declercq 
Journal of Investigative Dermatology 
Volume 137, Issue 2, Pages (February 2017)
DOI: /j.jid
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2. Figure 1
K5-ΔNp63αtg/tg mice have a disorganized hair coat and develop epidermal cysts on the tail. (a) Gross morphology at P21 reveals that K5-ΔNp63αtg/tg mice are smaller than wild-type mice and have a disorganized coat. K5-ΔNp63αtg/+ mice have an intermediate phenotype. (b) Western blot analysis of epidermal lysates of three wild-type, K5-ΔNp63αtg/+, and K5-ΔNp63αtg/tg mice using α-myc, α-p63, and α-β-tubulin antibodies. Total levels of ΔNp63α expression were quantified and normalized to β-tubulin; relative expression ratios are listed. (c) Scanning electronic microscopic analysis reveals bended hair tips in K5-ΔNp63αtg/tg mice. Scale bars represent 100 μm. (d) Gross morphology of epidermal cysts on the tail of K5-ΔNp63αtg/tg mice. Hematoxylin and eosin staining of the cross section of the tail skin containing bump reveals the presence of a cystic lesion lined by cornified epithelium containing lamellated keratin. Scale bar represents 100 μm. K5, keratin 5; WT, wild type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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3. Figure 2
K5-ΔNp63αtg/tg mice show epidermal hyperplasia and increased numbers of CD34+ hair follicle stem cells. (a) H&E staining on sagittal back skin sections reveals epidermal hyperplasia in K5-ΔNp63αtg/tg mice. (b) Flow cytometry analysis using α-CD34 antibodies on isolated keratinocytes from adult K5-ΔNp63αtg/tg and wild-type mice shows a higher percentage of CD34+ cells in transgenic animals. (c) Quantification of (a) and (b): scatter plots present mean + SEM; values were compared using a two-tailed unpaired Student t-test, *P < 0.05, ***P < (d) IHC analysis using α-myc, α-p63, α-Ki-67, α-keratin 10, α-loricrin (lor), and α-CD45 antibodies reveals expression of transgenic ΔNp63α, normal expression patterns of proliferation and differentiation markers, and absence of infiltrating immune cells in K5-ΔNp63αtg/tg mice. Scale bars represent 50 μm. H&E, hematoxylin and eosin; IHC, immunohistochemistry; K5, keratin 5; SEM, standard error of the mean; WT, wild type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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4. Figure 3
K5-ΔNp63αtg/tg mice display biliary dysmorphogenesis resulting in cyst formation and bile duct adenoma. (a) Gross morphology of liver covered with fluid-filled cysts of a K5-ΔNp63αtg/tg mouse killed when the belly appeared swollen. (b) H&E staining of the cystic lesion of the K5-ΔNp63αtg/tg mouse; lower panel: inset in magnification of a hyperplastic bile duct shows a normal bile duct of wild-type mice. IHC analysis using α-myc, α-keratin 7, and α-keratin 14 antibodies of cystic lesions. Intracellular mucus (arrows) was demonstrated by periodic acid-Schiff (PAS) staining. (c) H&E staining of livers reveals dilatation of the biliary duct (arrow) adjacent to the portal vein (pv) in K5-ΔNp63αtg/tg compared with wild-type mice at week 8. IHC analysis using α-myc and α-p63 antibodies shows the presence of ΔNp63α in K5-ΔNp63αtg/tg bile ducts. A specific cytoplasmic (*) or extracellular (**) staining with α-myc and α-p63 antibodies. Scale bars represent 50 μm. H&E, hematoxylin and eosin; IHC, immunohistochemistry; K5, keratin 5; WT, wild type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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5. Figure 4
Epidermal ΔNp63α overexpression facilitates tumor initiation in a gene dosage-dependent manner during chemical carcinogenesis. Distributions of proportions of papilloma-free mice of DMBA/TPA experiments were compared using the log-rank test in (a); numbers of papillomas measured in a 13-week (b) and a 6-week (c) time period were analyzed as repeated measurements using REML as implemented in Genstat (29), by fitting a autoregressive correlation model (AR1) to the log-transformed counts, followed by an F-test; means ± SEM are represented. The log value and tumor count are depicted respectively on the left and right sides of the Y-axis in (b) and (c), *P < 0.05, **P < 0.01, ***P < For (a)–(c): wild-type, n = 16; K5-ΔNp63αtg/+, n = 7; K5-ΔNp63αtg/tg, n = 14; (d) RFLP analysis of the Hras G61L mutation using XbaI digestion on amplified Hras exon 2 from DNA purified from wild-type and K5-ΔNp63αtg/tg papillomas and normal skin. Samples from five mice from each genotype were used for RFLP analysis. As tumor samples were not microdissected, weak cleavage band signals can be the result of too much accessory nontumor DNA in the sample. Note that in the K5-ΔNp63αtg/tg samples all tumors were positive for the G61L HRas mutation. (e) Gross morphology at week 25 of DMBA/TPA protocol (arrow point to SCC). (f) Distributions of proportions of tumor-free mice of DMBA experiment were compared using the log-rank test. (g) The number of SCCs were counted 4 months after ending the DMBA treatment protocol and compared using the Mann-Whitney U-test. Means ± SEM are represented. *P < For (f), (g): wild-type, n = 6; K5-ΔNp63αtg/tg, n = 9. (h) Gross morphology 4 months after the end of the DMBA treatment protocol (arrows point to SCC). DMBA/TPA, 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate; H&E, hematoxylin and eosin; IHC, immunohistochemistry; K5, keratin 5; REML, restriction maximum likelihood; RFLP, restriction fragment length polymorphism; SCC, squamous cell carcinoma; SEM, standard error of the mean.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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6. Figure 5
No difference in DMBA-induced keratinocyte apoptosis in K5-ΔNp63αtg/tg compared with wild-type mice. (a) IHC analysis using an α-active caspase-3 antibody on dorsal skin sections 24 hours and 72 hours after DMBA treatment (single dose: 25 μg in 200 μl acetone). Scale bars represent 50 μm. (b) Quantification of active casp-3 positive cells per mm epidermis in K5-ΔNp63αtg/tg and wild-type mice, DMBA-treated versus acetone control. The graph presents mean ± SEM; values were compared using two-way analysis of variance, revealing nonsignificant interaction (P = ). Wild-type, acetone, n = 6; wild-type, DMBA (24 hours), n = 8; wild-type, DMBA (72 hours), n = 14; K5-ΔNp63αtg/tg, acetone, n = 8; K5-ΔNp63αtg/tg, DMBA (24 hours), n = 10; K5-ΔNp63αtg/tg, DMBA (72 hours), n = 12. DMBA, 7,12-dimethylbenz[a]anthracene; IHC, immunohistochemistry; K5, keratin 5; SEM, standard error of the mean; WT, wild type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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7. Figure 6
Keratinocytes derived from K5-ΔNp63αtg/tg mice show a delay in cellular senescence. (a) Keratinocyte cultures derived from K5-ΔNp63αtg/tg and wild-type mice imaged by phase contrast microscopy at different passages (P0–2). (b) 5-ethynyl-2'-deoxy uridine (EdU) labeling of K5-ΔNp63αtg/tg and wild-type keratinocytes measured by flow cytometry at each passage. (c) Staining for senescence-associated β-galactosidase activity of K5-ΔNp63αtg/tg and wild-type keratinocytes at P2. (d) Western blot analysis of keratinocyte culture lysates at each passage using α-p63, α-p16Ink4a, α-p19Arf, α-Lsh, α-Sirt1, α-p53, α-p21, and α-β-tubulin antibodies. Scale bars represent 25 μm. EdU, 5-ethynyl-2'-deoxy uridine; K5, keratin 5; WT, wild type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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