1. Requirement of Zinc Transporter SLC39A7/ZIP7 for Dermal Development to Fine-Tune Endoplasmic Reticulum Function by Regulating Protein Disulfide Isomerase 
Bum-Ho Bin, Jinhyuk Bhin, Juyeon Seo, Se-Young Kim, Eunyoung Lee, Kyuhee Park, Dong-Hwa Choi, Teruhisa Takagishi, Takafumi Hara, Daehee Hwang, Haruhiko Koseki, Yoshinobu Asada, Shinji Shimoda, Kenji Mishima, Toshiyuki Fukada 
Journal of Investigative Dermatology 
Volume 137, Issue 8, Pages (August 2017)
DOI: /j.jid
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2. Figure 1
Zip7Col1 mice have impaired dermal development. (a) Five-week-old female wild-type (WT) and Zip7Col1 mice. The right panel indicates the body weight curves (n = 5 for each; *P < 0.05). (b) Zip7Col1 mice developed thin dermis. Scale bars = 200 μm. Double-headed arrows indicate dermis thickness. (c) The thickness of the Azan-stained dermis quantified by light microscopy. Five different parts of two skin specimens derived from each mouse were analyzed (***P < 0.001). M, muscle; S, Subcutis; Zip, zinc-regulation transporter-/iron-regulation transporter-like protein.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

3. Figure 2
ZIP7 is crucial for maintenance of hMSCs. (a) Cell growth after the siZIP7 treatment. The data are presented as mean ± SD of three independent experiments (***P < 0.001). (b) The siZIP7 treatment disturbs the differentiation of hMSCs into fibrogenic. hMSCs were differentiated by culturing in each differentiation medium for 3 weeks, and subsequently stained. Each siRNA was treated at every 4 days. Scale bars = 100 μm. The images of the stained cells from three independent experiments are shown. (c) The relative intensity of stained cells was measured using the ImageJ software ( and used for statistical analysis. The data are presented as mean ± SD of three independent experiments (***P < 0.001). hMSC, human mesenchymal stem cell; SD, standard deviation; siRNA, small interfering RNA; ZIP, zinc-regulation transporter-/iron-regulation transporter-like protein.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

4. Figure 3
ZIP7 is essential for ER functions. (a) Scatter plot representing the expression values (log2 scale) of normal (WT) versus ZIP7-KD (ZIP7 KD) hMSCs. Red dots indicate the upregulated genes and blue dots indicate the downregulated genes in ZIP7-KD hMSCs. Black dots indicate the genes not affected by ZIP7-KD. (b) Cellular processes represented by the upregulated (red bar) and downregulated (blue bar) genes in ZIP7-KD hMSCs. Green dotted line indicates the threshold of enrichment (P-value = 0.01). (c) Top 20 upregulated genes involved in ER stress response and apoptosis. (d and e) Quantification of mRNA levels by real-time PCR clarifies each changed gene in the siZIP7-treated hMSCs, related to (d) ER stress and (e) apoptosis. The data were normalized to the GAPDH expression. Data are presented as mean ± SD of three separate experiments (***P < 0.001). ER, endoplasmic reticulum; hMSC, human mesenchymal stem cell; KD, knockdown; SD, standard deviation; WT, wild-type; ZIP, zinc-regulation transporter-/iron-regulation transporter-like protein.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

5. Figure 4
ZIP7 is located on the ER. (a) Cellular location represented by the upregulated (red bar) and downregulated (blue bar) genes in ZIP7-KD hMSCs. Green dotted line indicates the threshold of enrichment (P-value = 0.01). (b) Cellular distribution of ZIP7 in hMSCs. Cells were transfected with plasmids encoding V5-tag, and stained with anti-V5 antibody together with either anti-BIP and TGN antibodies, or Golgi and ER trackers. Scale bars = 50 μm. ER, endoplasmic reticulum; hMSC, human mesenchymal stem cell; KD, knockdown; ZIP, zinc-regulation transporter-/iron-regulation transporter-like protein.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

6. Figure 5
ZIP7 is involved in the protein disulfide isomerase activity. (a) Western blot analysis reveals the elevation of aggregates of PDI in the siZIP7-treated hMSCs. (b) Zinc level was observed by Fluozin-3 staining. hMSCs were treated with each siRNA for 3 days. Fluozin-3 and ER tracker were added to the cells for 15 minutes before harvesting. Data were collected for three independent experiments (*P < 0.05). Scale bars = 25 μm. (c) Recombinant PDI proteins were treated with the indicated concentration of ZnSO4 for 1 hour, and then subjected to SDS-PAGE. (d and e) The PDI activity assay was performed according to the Materials and Methods section. Data are presented as mean ± SD of three separate experiments (***P < 0.001, *P < 0.05). ER, endoplasmic reticulum; hMSC, human mesenchymal stem cell; IB, immunoblotting; PDI, protein disulfide isomerase; SD, standard deviation; siRNA, small interfering RNA; ZIP, zinc-regulation transporter-/iron-regulation transporter-like protein.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

7. Figure 6
Models for the involvement of ZIP7 in ER function. When ZIP7 is dysregulated (right), the luminal zinc level may be elevated, which would induce zinc-dependent aggregation of PDIs. Therefore, protein folding and disulfide bond formation would proceed aberrantly, leading to the unfolded protein responses. ER, endoplasmic reticulum; KO, knockout; PDI, protein disulfide isomerase; UPR, unfolded protein response; ZIP, zinc-regulation transporter-/iron-regulation transporter-like protein.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions