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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 protects from cell death in stress situations in vitro. MiaPaCa2 cells were transfected with siCtrl or two different siNupr1 for 48 h. Cells were then incubated with thapsigargin, brefeldin A, or tunicamycin at 1 μM, for another 24 h in the presence or not of Z-VAD-FMK 10 μM or Nec-1 (40 μM). LDH release was measured (A); data values were normalized to the untreated siCtrl. In transfected MiaPaCa2 cells, flow cytometry analysis of annexin-V and PI staining following 24 h of treatment with thapsigargin, brefeldin A, or tunicamycin, showing % of PI-positive cells (B), annexin V-positive cells (C) and a representative experiment of the dot plot profile of cells (D). Data are means of triplicates ± SEM. For each treatment, statistically significant differences (*p < 0.05, **p < 0.01, ***p < 0.001) compared to siCtrl with same treatment conditions (^#p < 0.05, ^##p < 0.01, ^###p < 0.001) compared to non-pretreated cells with Z-VAD-FMK or Nec-1 with same ER-stressor treatment. Three Panc-1 control clones (with wild-type NUPR1) and 6 Nupr1 knockout clones, developed using CRISPR-Cas9 technology, were cultured in DMEM and incubated with thapsigargin, brefeldin A, or tunicamycin at 1 μM for 24 h. Flow cytometry analysis was carried out after Annexin-V and PI staining. The percentage of PI-positive cells (E), annexin V-positive cells (F) and a representative experiment of the dot plot profile of cells was showed (G). Data are means of triplicates ± SEM of 3 control and 6 Nupr1 knockout clones. For each treatment, statistically significant differences (*p < 0.05, **p < 0.01, ***p < 0.001) from control clones are shown.