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. 2017 Oct 27;7:14199. doi: 10.1038/s41598-017-14230-1

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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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α1D protein regulated cytosolic Ca²⁺ concentration by inhibiting NCX. (A) left, the schema represents NCX working in its forward mode driving Na⁺ entry and Ca²⁺ efflux. Middle, Histograms showing the effect of 10 µM SEA0400 or 30 µM KBR7943 on F340/F380 ratios. Results are normalized to CT conditions and are expressed as mean ± S.E.M. *Significantly different from control (p = 0.05, N = 3, n = 12, Kruskal-Wallis one way analysis of variance. Right, Representative cropped Western blot of NCX1 and NCX3 in HCT116 colon cancer cells (N = 3). Full-length Western blots are included in the Supplementary Fig. 6. (B) Histograms showing the effect of 10 µM SEA0400 on TG slope, the TG area, the TG peak Ca²⁺ responses and the TG relaxation slope. Results are normalized to CT conditions and are expressed as mean ± S.E.M. *Significantly different from control (p = 0.001, N = 3, n = 12, Mann Whitney test). (C) Time dependent measurements of [Ca²⁺]_c in the presence of 140 mM (Na140) or 10 mM (Na10) of external Na⁺ concentration. The schema represents NCX working in its reverse mode driving Na⁺ efflux and Ca²⁺ entry. Histograms showing the effect of reducing external Na⁺ concentration to 10 mM on the amplitude and on the frequency of Ca²⁺ responses. Results are expressed as mean ± S.E.M. (D) Time dependent measurements of [Ca²⁺]_c in the presence of 140 mM or 10 mM of external Na⁺ concentration in control cells or in cells that have been silenced for significant differences between control and1D. Histograms showing the effect of silencing significant differences between control and1D (si significant differences between control and1D#1) on the amplitude and on the frequency of Ca²⁺ responses. Results are expressed as mean ± S.E.M. *Significantly different from control (amplitude: p = 0.001, N = 5, n = 77; N = 6, n = 94, Mann Whitney test and for frequency: p = 0.001, N = 5, n = 53; N = 6, n = 81, Mann Whitney test). (E), examples of membrane potentials recorded in one control HCT116 cell and in one HCT116 cell that have been silenced for α1D using the patch clamp technique in current clamp mode. Membrane potentials were recorded in Na140 or Na10 solutions. Histogram showing membrane potential variations between Na140 and Na10 conditions in control and significant differences between control and1D cells. Results are expressed as mean, bars, SEM. *Significantly different from control (p = 0.005, N = 3, n = 7 si-control; N = 3, n = 6 si significant differences between control and1D#1, Mann Whitney test). ND: not determined. F. Effect of silencing significant differences between control and1D in presence or not of 1 µM SEA0400 on migration of HCT116 cells (wound-healing cell migration assays). Results are normalized to si control conditions and results are expressed as mean ± S.E.M. *Significantly different from control (Si-Control with SEA0400; si significant differences between control and1D; si significant differences between control and1D with SEA0400, N = 4, n = 14).