Mechanism Underlying ATP Release in Human Epidermal Keratinocytes

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Mechanism Underlying ATP Release in Human Epidermal Keratinocytes Kaori Inoue, Ryohei Komatsu, Yoshio Imura, Kayoko Fujishita, Keisuke Shibata, Yoshinori Moriyama, Schuichi Koizumi Journal of Investigative Dermatology Volume 134, Issue 5, Pages 1465-1468 (May 2014) DOI: 10.1038/jid.2013.516 Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Expression of vesicular nucleotide transporter (VNUT) and VNUT-dependent adenosine triphosphate (ATP) release. (a) (i) Immunohistochemical and (ii) immunocytochemical staining of a frozen section of human skin and normal human epidermal keratinocytes (NHEKs), respectively, with VNUT-antibody. (iii) VNUT section was co-stained with anti-transglutaminase and DAPI. (iii) For western blot analysis, the antibody that specifically recognizes VNUT protein detected major bands at 65kDa and 68kDa. (b) Transfection with VNUT siRNA for 72hours (n=3, black bars) significantly decreased ionomycin-evoked ATP release versus scrambled (scr) siRNA-transfection (white bars) at concentrations of 2 and 5μM ionomycin. ***P<0.001 vs. basal, ###P<0.001 vs. scr siRNA. Scale bars=200μm (a (i) VNUT), 50μm (a (i) high magnification), 10μm (a (ii)). Journal of Investigative Dermatology 2014 134, 1465-1468DOI: (10.1038/jid.2013.516) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Mechanical stimulation (MS)-evoked ATP release and ATP-induced ATP release from NHEKs. (a) Quinacrine-labeled fluorescent and bright-field images of NHEKs. Distance from MS cell (cell 1) is shown. (b) Left, typical quinacrine-labeled fluorescent images of cell 1, 2, and 5. Right upper: typical sequential images of single quinacrine fluorescence (vesicle #1–#3, shown as squares in left panels) in response to MS. Right lower: time-course of MS-induced quinacrine-quenching of individual vesicles (typical 2 vesicles) in time-lapse recording for the indicated period. Arrows show MS of cell 1. (c) Histogram of frequency of exocytosis from individual vesicles of MS cell (cell 1) and adjacent cells (cells 2 and 5) over time. NHEKs, normal human epidermal keratinocytes. Journal of Investigative Dermatology 2014 134, 1465-1468DOI: (10.1038/jid.2013.516) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions