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. 2018 Mar 23;8:5118. doi: 10.1038/s41598-018-23175-y

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© The Author(s) 2018

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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Irp1/Irp2 ablation induces cellular iron deficient, but not deficient in mitochondrial labile iron pool (LIP). (a) The purity of extra-mitochondrial (Extra-mito) and mitochondrial (Mito) fractions was analysed by Western blot. Xod and CytC were used as extra-mito and mito markers, respectively. A representative image set is presented. The purity of Extra-mito and Mito fractions was quantified by calculating the relative percentage of Xod or CytC in Extra-mito and Mito fractions after normalisation with total proteins (bottom panel). (b) Extra-mito and Mito total iron levels in mouse embryonic fibroblasts (MEFs) of Irp1^−/−, Irp2^−/−, and wild type (WT) measured by Ferrozine assays. (c) Levels of cytosolic (Cyt) and Mito LIP in MEFs of Irp1^−/−, Irp2^−/−, and WT measured by Calcein-AM and Rhodamine B-[(1, 10-phenanthroline-5-yl)-aminocarbonyl] benzyl ester (RPA), respectively. Values represent mean ± SEM (n = 8). A one-way ANOVA was performed. ***p < 0.001 compared with WT.