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. 2018 Nov 19;8:16999. doi: 10.1038/s41598-018-35020-3

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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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Nupr1 deficiency leads to a mitochondrial membrane potential disruption inducing ROS overproduction. Flow cytometry analysis of MiaPaCa2 cells transfected with siCtrl, siNUPR1-1, or siNUPR1-2 for 72 h was carried out using MITO-ID for analysis of the mitochondrial membrane potential (A). MITO-ID was also applied to three clones of Panc-1 control cells (with wild-type NUPR1) and six clones of Nupr1 knockout cells, developed by CRISPR-Cas9 technology. Data are means of triplicates ± SEM of the 3 control and 6 knockout clones (B). ROS production was detected using MitoSOX Red by flow cytometry analysis in the experimental cell groups as described above (C,D). Representative images of the fluorescence microscopy of both ROS indicators are shown (C,D). Data are means of triplicates ± SEM. Statistically significant differences from siCtrl (*p < 0.05, **p < 0.01, ***p < 0.001) was shown.