1. An Acrodermatitis Enteropathica-Associated Zn Transporter, ZIP4, Regulates Human Epidermal Homeostasis 
Bum-Ho Bin, Jinhyuk Bhin, Nan-Hyung Kim, Su-Hyon Lee, Haeng-Sun Jung, Juyeon Seo, Dae-Kyum Kim, Daehee Hwang, Toshiyuki Fukada, Ai-Young Lee, Tae Ryong Lee, Eun-Gyung Cho 
Journal of Investigative Dermatology 
Volume 137, Issue 4, Pages (April 2017)
DOI: /j.jid
Copyright © 2016 The Authors Terms and Conditions

2. Figure 1
ZIP4, a major Zn transporter in human epidermis, regulates epidermal Zn homeostasis. (a) Expression of the MT protein in human epidermis. Scale bars = 50 μm. (b) Intracellular Zn levels were seen by FluoZin-3 staining. Scale bars = 50 μm. (c) Human epidermal keratinocytes were incubated with conditional medium containing either TPEN (5 μM) alone or TPEN (5 μM) with the indicated metal compounds (each 5 μM) for 72 hours. Scale bars = 100 μm. (d) The expression of Zn transporters was analyzed with quantitative reverse transcriptase-PCR. *P < (e) Expression levels of the ZIP4 protein. (f) Expression levels of the ZIP4 protein in cells harboring either control shRNA or shZIP4. (g) Intracellular Zn levels were seen by FluoZin-3 staining. Relative intensity (bottom). Scale bars = 50 μm; *P < 0.05. (h) Measurement of Zn level with inductively coupled plasma atomic emission spectroscopy. ***P < 0.005.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

3. Figure 2
ZIP4 knockdown disturbs the formation of the epidermis in a reconstituted human skin model. (a, b) Human epidermal keratinocytes were treated with siControl or siZIP4 for 3 days. The expression of ZIP4 was analyzed with quantitative reverse transcriptase-PCR (a) or with western blot analysis (b). (c) ZIP4 KD disturbs epidermal stratification in a human skin equivalent model. Scale bars = 400 μm. (d) ZIP4 KD reduces the expression of filaggrin, involucrin, and keratin 14 in a human skin equivalent model. Scale bars = 400 μm. (e) Immunohistochemistry for proliferating cell nuclear antigen reveals that ZIP4 KD disturbs the proper formation of the epidermis. Scale bars = 100 μm. (f) Human epidermal keratinocytes were treated with siRNA for 48 hours and then cultured with medium containing 1.8 mM Ca2+ for 4 days. Cells were visualized with an inverted microscope. Boxed areas are magnified. Scale bars = 50 μm.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

4. Figure 3
Zn and ZIP4 are essential for p63 activity during epidermal differentiation. (a) Immunohistochemical staining for p63 found that ZIP4 KD disturbs the proper formation of the epidermis. Scale bars = 100 μm; Vertical bar indicates epidermal thickness. Asterisk indicates suprabasal layer. (b) H1299 cells stably expressing the ΔNp63 protein were transfected with siZIP4. Forty-eight hours after transfection, the cells were further transfected with PG13-Luc for 24 hours, and a luciferase assay was performed. ***P < (c) The expression levels of ADA, CCND3, and CHUK in human epidermal keratinocytes were analyzed with quantitative reverse transcriptase-PCR. *P < (d) Human epidermal keratinocytes were treated with 5 μM TPEN in combination with 5 μM ZnSO4 for 24 hours, after which mRNA was isolated. The expression levels of each gene in human epidermal keratinocytes were analyzed with quantitative reverse transcriptase-PCR. ***P <
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

5. Figure 4
Cys205 in the Zn-binding motif is critical for the Zn-dependent functional activity of ΔNp63. (a) Models of the ΔNp63Mut-4S protein. (b) The expression of ΔNp63Mut-4S in HeLa cells was analyzed by western blotting 24 hours after DOX incubation. (c) Nuclear localization of the ΔNp63Mut-4S protein. Scale bars = 25 μm. (d) Models of the mutated p63 proteins. (e) The nuclear localization of mutant p63 proteins. Scale bars = 25 μm. (f) Expression of the C205S (ΔNp63Mut-1S) mutant protein. (g) H1299 cells were transfected with each ΔNp63 construct and PG13-Luc for 24 hours, after which a luciferase assay was performed. ***P < (h) Posttranslational degradation of ΔNp63WT under Zn deficiency. 293T cells were analyzed after 10 μM CHX treatment in combination with 2 μM TPEN for 24 hours. The relative expression level (right). Data are representative of two independent experiments.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

6. Figure 5
ZIP4 is enriched in the hyperproliferative rete ridge area of psoriatic skin. (a) Normal areas (N) and (b) psoriatic skin lesions (P) in human skin specimens obtained from two psoriasis patients (#1 and #2) were stained with anti-ZIP4 antibody (green); nuclei were stained with DAPI (blue). Arrowheads indicate the apparent expression of ZIP4. The insets are magnified. Scale bars = 400 μm.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions