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. 2020 Feb 21;10:3163. doi: 10.1038/s41598-020-59991-4

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© The Author(s) 2020

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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Iron handling in P19 cells under various conditions of extracellular iron supplementation. Cells were cultured either in non-supplemented medium (−Fe) or iron-supplemented medium (+Fe) for at least 5–7 days before iron supplementation withdrawal and an additional 1 (1h-Fe), 2 (2h-Fe), 4 (4h-Fe) or 24 (24h-Fe) hours of culture in non-supplemented medium. (a) Total cellular iron content was measured by ICP-MS and normalized to total amount of protein. Data are the mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001): −Fe, n = 4; +Fe, n = 9; 1h-Fe, n = 3; 2h-Fe, n = 3; 4h-Fe, n = 3; 24h-Fe, n = 20. (b) Protein lysates from P19 cells were examined by western blot, probed with α-FPN 1 (top panel) and α-GAPDH (bottom panel). Approximate MW is indicated in the left margin. Full-length blots are presented in Supplementary Fig. S1. (c) Similar immunoblots were also probed with α-TfRc (top panel) and α-GAPDH (bottom panel). (d) The signal intensity of each TfRc band was normalized to the corresponding GAPDH band, revealing little or no change in the level of TfRc. Full-length blots are presented in Supplementary Fig. S2.