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. 2017 Nov 9;7:15137. doi: 10.1038/s41598-017-15085-2

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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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Microarray analysis revealed an 8.5-fold increase in gene expression of transferrin receptor (Tfrc) in the placentas of the zinc-deficient dams (a). This increase was validated and confirmed in an independent cohort of placental samples using qPCR (b). qPCR was also used to measure Tfrc expression within kidney tissue at GD18.5 and was also increased in tissue collected from zinc-deficient dams (c). Immunohistochemical analysis of Tfrc protein showed localisation to the apical surface of the trophoblast cells within the labyrinth zone (LZ) of the placenta (d). Tfrc protein expression was analysed using Western blot and compared to β-actin expression (e); Tfrc band at ~100 kD and β-actin at ~42 kD, (full image in supplementary information). This revealed an increased expression of Tfrc protein in placentas from zinc-deficient dams (f). Transferrin (Trf) gene expression within the placenta and kidney was also quantified by qPCR but did not differ between the two diet groups (g,h). Data are median and interquartile range (n = 19 and 12 placentas [a]; 50 and 32 placentas [b and g]; 12 and 11 kidneys [c and h] and 12 and 10 placentas [e and f] from 12 zinc-replete and 11 zinc-deficient dams, respectively). Statistical significance was determined using Mann-Whitney Test. *P < 0.05, ***P < 0.001. JZ: junctional zone.