1. ERBB2 Is Essential for the Growth of Chemically Induced Skin Tumors in Mice 
Maik Dahlhoff, Sukalp Muzumdar, Matthias Schäfer, Marlon R. Schneider 
Journal of Investigative Dermatology 
Volume 137, Issue 4, Pages (April 2017)
DOI: /j.jid
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2. Figure 1
ERBB2 is not essential for the development and homeostasis of mouse skin. (a) Schematic drawing of the floxed Erbb2 allele and the recombined (=deleted) allele after cre recombinase-mediated removal of exons 10–12. The red arrowheads indicate loxP sites, and the arrows indicate the location of PCR primers. (b) PCR demonstrating the existence of a deleted Erbb2 allele exclusively in the skin of K5Cre;Erbb2del mice; amplification of Gapdh is used as a control. In, small intestine; Ki, kidney; Li, liver; Mu, muscle; Sk, back skin; Sp, spleen; Te, testes. (c) Western blot analysis demonstrated loss of ERBB2 in the skin but unchanged receptor levels in the lung of K5Cre;Erbb2del mice. GAPDH is shown as a loading control. (d) Immunohistochemistry revealing normal levels of ERBB2 in the tail skin epidermis of control mice and the absence of ERBB2 in the tail skin epidermis of K5Cre;Erbb2del mice. Bars in (d) represent 100 μm. ERBB, erythroblastic leukemia viral oncogene; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; K5, keratin 5.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

3. Figure 2
Western blot analysis of isolated epidermis from newborn Erbb2del and control mice. The total and the phosphorylated levels of the receptors EGFR, ERBB2, and ERBB3 (a) and of the downstream signaling proteins MAPK1/2, MAPK14, and AKT (b) are shown. Each lane depicts protein taken from a single newborn animal (all females). The densitometric quantification and normalization to GAPDH are shown at the bottom. n = 4, *P < 0.05, **P < 0.01, ***P < AKT, Rac-alpha serine/threonine-protein kinase; ERBB, erythroblastic leukemia viral oncogene; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MAPK, mitogen-activated protein kinase.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

4. Figure 3
Loss of ERBB2 impairs full-thickness wound healing. (a) Schematic drawing of a 5-day wound (5dw). The wound epithelium (WE) is highlighted in red, the outer thickened epithelium (OTE) in blue, and the granulation tissue (G) in purple. Measurement of the length of the WE and OTE is indicated as a black line. (b) Pictures of a 5dw of a control (left) and an Erbb2del mouse (right) generated with a 5-mm biopsy punch. The wound epithelium is highlighted by a dotted line. (c–h) Wound closure (c), wound diameter (d), length (e), thickness (f), area (g), number of cells (h), and proliferation rate of keratinocytes (i) in the wound epidermis of control and Erbb2del mice 5 days after wounding. n = 10–11 mice, with one anterior and one posterior wound per mouse. (j–m) Length (j), thickness (k), area (l), and proliferation rate of keratinocytes (m) in the wound epidermis of control and Erbb2del mice 10 days after wounding. n = 9 mice, with one anterior and one posterior wound per mouse. Data were analyzed with the Mann-Whitney U-test and error bars represent standard deviation. Scale bar in (b) represents 100 μm. *P < 0.05, **P < 0.01, ***P < D, dermis; E, epidermis; ES, eschar.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

5. Figure 4
ERBB2 is important for cell proliferation and migration. Primary keratinocytes were isolated from a pool of six Erbb2del and four control mice. All analyses shown below were performed using six replicates of these pooled keratinocytes. (a) Scratch assay of primary keratinocytes shows that the gap closure is significantly delayed at 48 hours and 72 hours in Erbb2del keratinocytes. (b) BrdU assay of primary keratinocytes reveals a significantly decreased proliferation in Erbb2del keratinocytes. Green = BrdU-positive; red = propidium iodide (all nuclei). (c) Differentiation markers K5 and K6 are unchanged, but K10 and filaggrin expression is significantly decreased in Erbb2del keratinocytes. Data were analyzed with Student’s t-test and error bars represent SEM. *P < 0.05, **P < 0.01, ***P < Scale bars in (a) represent 0.5 mm and in (b) 200 μm. FLG, filaggrin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; K5, keratin 5; K6, keratin 6; K10, keratin 10; SEM, standard error of the mean.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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6. Figure 5
ERBB2 is necessary for skin tumor development. Analysis of tumor incidence (a), tumor number/mouse (b), mean tumor size/mouse (c), and tumor burden (d) in Erbb2del (n = 19) and control (n = 24) littermates. (e) Representative photographs of the back skin of Erbb2del and control animals at the end of the study. (f) Analysis of H&E-stained histological sections revealed a weak, diffuse epithelial hyperproliferation in Erbb2del mice in contrast to full-grown papillomas in control littermates. (g) Ki67 immunohistochemistry revealed impaired cell proliferation in Erbb2del tumors (right panel) compared with control tumors (left panel). Data in (b)–(d) are means ± SEM. Two-way ANOVA of data in (a)–(d) revealed a statistically significant difference between genotypes, P <  Scale bars represent 500 μm in (f) and (g) and 50 μm in the insets. ANOVA, analysis of variance; DMBA, 7,12-dimethylbenz[a]anthracene; H&E, hematoxylin and eosin; SEM, standard error of the mean.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

7. Figure 6
Deletion of ERBB2 reduces TPA-induced epidermal hyperproliferation. (a) Immunohistochemistry revealed a lower number of Ki67-positive nuclei in the back skin epidermis of Erbb2del mice compared with control animals at 24 hours and 48 hours after TPA application. Analysis of epidermal thickness (b) and proliferation (c) at both time points. Data were analyzed with Student’s t-test. Error bars represent SEM. P < 0.05 was considered significant. **P < n = 6/group (24 hours) and n = 5/group (48 hours). Scale bars represent 50 μm. SEM, standard error of the mean; TPA, 12-O-tetradecanoylphorbol-13-acetate.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions