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. 2017 Jun 27;7:4265. doi: 10.1038/s41598-017-04069-x

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Evaluation of the induced expression of hepcidin and reduced serum iron concentration upon A. hydrophila DNA stimulation. (A) Detection of the steady state level of hepcidin mRNA in liver upon stimulation with various concentrations of A. hydrophila DNA (0.5, 1, 3, and 5 μg per fish) by Q-PCR. (B and C) Detection of the hepcidin protein level in serum upon stimulation with various concentrations of A. hydrophila DNA (0.5, 1, 3, and 5 μg per fish) by Western blot (B) and ELISA (C), and the gray value of the relative signal intensity (hepcidin/GAPDH) in Western blot analysis was calculated using ImageJ program and depicted in a bar graph. (D) Time-dependent upregulation of the steady state level of hepcidin mRNA in liver upon stimulation with A. hydrophila DNA (1 μg per fish) as determined by Q-PCR. (E) Time-dependent upregulation of the hepcidin protein in serum upon stimulation with A. hydrophila DNA (1 μg per fish) as determined by ELISA. (F) Reduced concentrations of serum iron stimulated by A. hydrophila DNA (0.5, 1, 3, and 5 μg per fish) were detected by ICP-MS. The control groups in each experiment (indicated as Control) received mock PBS. The steady state level of hepcidin mRNA in liver was calculated by 2^−ΔΔCT method normalized to β-actin. All data are representative of at least three independent experiments. A significant difference was detected between each experimental and control group. *p < 0.05, **p < 0.01. ns for not significant. The full-length blots are presented in Supplementary Fig. S4.