1. Transcription Factor MafB Coordinates Epidermal Keratinocyte Differentiation 
Masashi Miyai, Michito Hamada, Takashi Moriguchi, Junichiro Hiruma, Akiyo Kamitani-Kawamoto, Hajime Watanabe, Mariko Hara- Chikuma, Kenzo Takahashi, Satoru Takahashi, Kohsuke Kataoka 
Journal of Investigative Dermatology 
Volume 136, Issue 9, Pages (September 2016)
DOI: /j.jid
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2. Figure 1
Expression of MafB in the epidermis of developing mouse embryos. (a–f) Bright-field microscopic view (a, c, e) and immunostaining of MafB (magenta) (b, d, f) for mouse dorsal skin sections at embryonic day (E) 13.5 (a, b), E15.5 (c, d), and E18.5 (e, f). Nuclei were visualized with DAPI (blue). Data are representatives of three (E13.5 and E15.5) or ten (E18.5) biological replicates. Scale bars = 20 μm. (g–i) E18.5 skin sections were coimmunostained for MafB (green) and Krt5 (g), Krt10 (h), or Ki67 (i) (magenta). Dotted lines mark the basement membrane and the edge of the CL. An asterisk in (h) is a nonspecific signal in the CL. Data are representatives of ten (g, h) and five (i) independent experiments. Scale bars = 20 μm. (j) Schematic representation of MafB expression in the epidermis. Keratinocytes expressing MafB during the differentiation process are labeled in green. CL, cornified layer; Krt, keratin.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

3. Figure 2
Induction of the MAFB gene promoter during keratinocyte differentiation. (a) Quantitative real-time reverse transcriptase-PCR (Q-RT-PCR) analysis. RNAs were isolated at 80% confluence, 100% confluence, 2 days after confluence (120%), and 2 days after confluence followed by 1.2 mM CaCl2 treatment. Results are the means ± SEM (n = 3). (b) Computational analysis of MafB locus using the ECR browser ( Blue and yellow regions represent the open reading frame and 5′/3′ noncoding regions, respectively. (c) Luciferase assay. Indicated reporter plasmids were transfected into keratinocytes at 80% confluency, and luciferase activity was measured after reaching 100% or 120% confluency or after CaCl2 treatment. Luciferase activity was expressed relative to the activity in cells that received pGL4.12 plasmid at 100%. Data represent the means ± SEM (n = 3). SEM, standard error of the mean.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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4. Figure 3
Impaired epidermal differentiation in MafB–/– mice. (a, b) Histology of dorsal skin from embryonic day (E) 18.5 MafB+/+ and MafB–/– mice. Scale bars = 20 μm. (c–f) Higher magnifications of the boxed areas in (a) and (b). (g, h) DAPI staining of E18.5 skin sections of MafB+/+ (g) and MafB–/– (h) mice. Dotted lines mark the basement membrane and the edge of the CL and arrowheads indicate nuclei in the CL. Scale bars = 20 μm. (i, j) Quantifications of thickness of the CL (i) and viable (basal: Ba, spinous: Sp, and granular: Gr) layers (j) of MafB+/+ (n = 5) and MafB–/– (n = 5) mice. *P < (k–n) Dye permeability assay performed on E17.5 (k, l) and E18.5 embryos (m, n). (o, p) CEs prepared from E18.5 MafB+/+ (o) and MafB–/– (p) skin. CL, cornified layer; CE, cornified envelope.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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5. Figure 4
Impaired basal to spinous cell differentiation of MafB–/– keratinocytes. (a–p) Sections from MafB+/+ and MafB–/– dorsal skin at embryonic day (E) 18.5 were stained for the indicated antibodies. Nuclei were counterstained with DAPI. Dotted lines mark the basement membrane, and asterisks indicate nonspecific signals in the CL. Arrowheads indicate nuclei in the CL. s, g, and c in (o) represent filaggrin signals in the spinous, granular, and cornified layers, respectively. Data are representatives of at least five independent experiments. Scale bars = 20 μm. (q) Immunoblot analysis. Protein extracts were prepared from two MafB+/+ and MafB–/– embryos, and were analyzed for immunoblotting with the indicated antibodies. GAPDH was used as a loading control. (r) Epidermal protein extracts prepared from two MafB+/+ and MafB–/– littermates at E18.5 were analyzed by immunoblot using an anti-filaggrin antibody. GAPDH was used as a loading control. CL, cornified layer; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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6. Figure 5
Microarray analysis. (a) Gene ontology terms for downregulated and upregulated genes in MafB+/+ versus MafB–/– epidermis at embryonic day (E) (b) Q-RT-PCR analysis in MafB+/+ (n = 3) and MafB–/– (n = 3) epidermis. Results are means ± SEM (n.s., not significant, *P < 0.05, **P < 0.01). (c) List of lipid metabolism-related genes that were upregulated in the MafB–/– epidermis. Genes that are downregulated in Klf4-, Gata3-, or Grhl3-knockout mice are highlighted. (d) Q-RT-PCR analysis of lipid metabolism-related genes (*P < 0.05). (e) Heat map representation of upregulated (magenta) and downregulated (green) genes in MafB–/– mice together with data from a microarray analysis of the Grhl3–/– epidermis. (f) Q-RT-PCR analysis of mRNAs for Gata3, Grhl3, and Klf4 (n.s., not significant, **P < 0.01). GO, gene ontology; Q-RT-PCR, quantitative real-time reverse transcriptase-PCR; SEM, standard error of the mean.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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7. Figure 6
Reduced expression of filaggrin and MAFB in AD and PS skin. (a–h) Sections of normal (a, c) and AD (b, d) skin from human biopsies were stained with anti-MAFB and anti-filaggrin antibodies. The merged images of MAFB (green) and filaggrin (red) (a, b) are shown. Nuclei were also visualized with DAPI (blue) (c, d). Arrowheads indicate representative MAFB-positive nuclei. (e–h) MAFB immunostaining (green) of normal (e) and AD (f) skin specimens was observed by confocal microscopy. Nuclei were counterstained with DAPI (blue). (g, h) Higher magnifications of the boxed areas in (e) and (f). Data are representatives of sections from five (normal) and five (AD) individuals. Scale bars = 50 μm. (i–l) Sections of nonpsoriatic area (i, k) and psoriatic lesion (j, l) of skin biopsies from an individual with PS were stained with anti-MAFB and anti-filaggrin antibodies. MAFB signals (red) (i, j) and merged image of MAFB (red) and filaggrin (green) (k, l) are shown. Nuclei were also visualized with DAPI (blue). Data are representatives of sections from two PS individuals. Scale bars = 10 μm. AD, atopic dermatitis; PS, psoriasis vulgaris.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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