1. Electrical Stimulation Enhances Epidermal Proliferation in Human Cutaneous Wounds by Modulating p53–SIVA1 Interaction 
Anil Sebastian, Syed A. Iqbal, James Colthurst, Susan W. Volk, Ardeshir Bayat 
Journal of Investigative Dermatology 
Volume 135, Issue 4, Pages (April 2015)
DOI: /jid
Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Maintainence of human skin explants in tissue culture. (a) Within the limited exposure to electrical stimulation (ES; 100 mV mm−1 every day for 30 min), ES explants had partial dermal wound closure compared with control explants, on day 16. (b) Immunohistochemical analysis of cytokeratin 10 on day 0 and day 16 explants, with and without ES. (c) Messenger RNA (mRNA) transcript quantification of cytokeratin 10. There was nearly a threefold increase in mRNA expression of cytokeratin 10 in day 16 ES explants compared with day 16 control explants. (d) Epidermal thickness was nearly twofold in day 16 ES explants compared with day 16 control explants, showing similarity of ES explants to normal skin. DC, direct current; DW, degenerate wave. Bar = 1 mm for (a) and 50 μm for (b).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Electrical stimulation (ES) upregulates proliferating cell nuclear antigen (PCNA), human double minute 2 (HDM2), and SIVA1 in human skin explants. Protein analysis of (a, b) PCNA, (a, c) Phospho-p53, (a, d) HDM2, and (a, e) SIVA1 on day 0 and day 16 skin explants, with and without ES. On day 16, PCNA, HDM2, and SIVA1 were significantly upregulated (P<0.05) in explants subjected to ES compared with control explants. However, there was a stable expression of phospho-53 (P>0.05). Immunohistochemical analysis (Supplementary Figure S3) also supported this observation. DC, direct current; DW, degenerate wave; NS, nonsignificant.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Proliferation was decreased in keratinocytes subjected to electrical stimulation (ES), post SIVA1 single-interferenceRNA (siRNA) transfection. (a) Western blotting showed upregulation of proliferating cell nuclear antigen (PCNA), human double minute 2 (HDM2), and SIVA1 expression post ES. (b) Densitometric analysis of band intensities in a showed significant increase in PCNA, HDM2, and SIVA1 (P<0.05). (c) Coimmunoprecipitation (IP) followed by western blotting of phospho-p53 and SIVA1. (d) Western blotting of keratinocytes subjected to SIVA1 siRNA transfection. (e) Densitometric analysis of band intensities in d showed the upregulation of HDM2 and downregulation of proliferation in keratinocytes subjected to ‘SIVA1 siRNA transfection+ES’ compared with siRNA-transfected keratinocytes. (f) Fold change in messengerRNA expression of ‘keratinocytes+ES’, ‘keratinocytes+SIVA1 siRNA’, and ‘keratinocytes+SIVA1 siRNA+ES’, compared with wild-type keratinocytes. NS represents nonsignificant. DC, direct current; DW, degenerate wave; IP, immunoprecipitation. Bar = 50 μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Upregulation of p53 in keratinocytes subjected to ‘SIVA1 single-interferenceRNA (siRNA) transfection+ES’. (a) Intracellular localization of SIVA1 and p53 by double immunolabeling. SIVA1 knocked-down keratinocytes post direct current (DW) electrical stimulation (ES) showed stronger signals of p53 accumulated in nuclear and perinuclear regions. (b) Phospho-p53 from nuclear lysate analyzed by western blotting using histone deacetylase (HDAC1) as the internal control. (c) Higher expression of human double minute 2 (HDM2) was observed in the cytoplasm and the nucleus of SIVA1 knocked-down keratinocytes post DW ES, compared with ‘keratinocytes+DW ES’. (d) Cell cycle analysis showed an increase of S phase in ‘keratinocytes+DW ES’ compared with wild-type keratinocytes. In ‘keratinocytes+SIVA1 siRNA+DW ES’, there was an increase in sub-G1 and decrease in G1 and S phases compared with ‘keratinocytes+DW ES’. DAPI, 4′,6-diamidino-2-phenylindole.
Journal of Investigative Dermatology  , DOI: ( /jid )
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6. Figure 5
Proliferation, phospho-p53–SIVA1 coexpression, and ternary complex interactions were upregulated on day 7 in healing epidermis treated with electrical stimulation (ES). (a) ES-treated wounds showed higher proliferation than non-ES-treated wounds until day 7. (b) Quantitative analysis of phospho-p53 and SIVA showed that the percentage of positive cells increased until day 7. (c) Analysis of coexpression of phospho-p53–SIVA1 and human double minute 2 (HDM2)-SIVA1 showed higher percentage of coexpression until day 3. On further days, there was higher downregulation of p53–SIVA1 coexpression in non-ES-treated wounds compared with ES-treated wounds. (d) Ternary complex on healing day 7. We suggest higher transcriptional repression activities of HDM2 in association with SIVA1 on p53 targets in ES-treated wounds than in non-ES-treated wounds. () Normal healing wounds; () ES-treated wounds. PCNA, proliferating cell nuclear antigen; Pho, phospho.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions