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. 2022 May 5;12:7334. doi: 10.1038/s41598-022-10925-2

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© The Author(s) 2022

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Potent induction of ZNT1 expression mediated by Zip5 is associated with Zip5 cytosolic variable loop. (A) Zinc content measured using ICP-MS analysis. Parental MDCK cells and MDCK cells stably expressing Zip4-HA, Zip5-HA, ZIP4-HA, or ZIP5-HA were cultured with 1.0 μg/mL Dox for 24 h and then collected for ICP-MS analyses (n = 3). (B) Immunoblotting performed using cell lysates prepared from the indicated cells cultured as in (A). (C) ZNT1 and MT expression induced by Zip4_TM3–4-HA mutant was higher than that induced by Zip4. Parental MDCK cells and MDCK cells stably expressing Zip4-HA or Zip4_TM3–4-HA were cultured with 1.0 μg/mL Dox for 24 h. (D) Immunofluorescence labeling for confirming enhanced cell-surface expression of ZNT1 in MDCK cells stably expressing Zip4_TM3–4-HA; the cells were cultured as in (C). (E) Zinc content, measured using ICP-MS analysis, in indicated MDCK cells cultured as in (C) (n = 3). In (B,C), tubulin was used as the loading control. Each experiment was performed at least thrice, and representative results from independent experiments are shown.