Induction of the UPR by NADD variants.

<p>(a) HUVECs were transduced by lentiviral particles containing either an ERSE- luciferase reporter or a luciferase construct under control of a CMV driven promoter as transduction efficiency control. The cells were stimulated for 8 hrs with 100 µM of different NADD as indicated. Equimolar concentrations of dopamine were also included to assess if dopamine induces the UPR. ERSE dependent luciferase activity was assessed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099298#s2" target="_blank">materials and methods</a> and normalized for constitutively expressed luciferase. The results are expressed as normalized ESRE luciferase activity relative to untreated (Med.) cells. Values represent mean ± SD from three independent experiments, *<i>P</i><0.05, **<i>P</i><0.01 vs. untreated control, <i>ns:</i> not significant. (b) HUVECs were treated with 100 µM NADD for 8 hrs. Cells that were not treated (Med.) served as control. Hereafter total RNA was isolated and the expression of BiP and GAPDH were quantitated by qPCR. The results are expressed as BiP mRNA levels, normalized for GAPDH mRNA expression and relative to cells that were not treated (Med), *<i>P</i><0.05, **<i>P</i><0.01 vs. untreated control, <i>ns</i>: not significant. (c) HUVECs were stimulated for 8 hrs with 100 µM of different NADD, or left untreated (Med). Hereafter protein extracts were made and phosphorylation of eIF2α was assessed by Western blotting. The blots were stripped and re-probed with antibodies directed against total eIF2α and β-actin to test for equal loading. The results of a representative blot are depicted, a total of 4 independent experiments were performed. (d) HUVECs were stimulated for 8 hrs with different NADD (100 µM) before total RNA was isolated. Cells that were not treated (Med.) served as control. Spliced and unspliced xbp-1 mRNA were detected by PCR. The spliced and unspliced amplification products differ by 26 bp. (e) Cells were transduced with lentiviral particle containing an ATF-6-driven luciferase reporter or a constitutively expressed luciferase construct. The cells were stimulated for 8 hrs with different NADD (100 µM). Luciferase activity was assessed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099298#s2" target="_blank">materials and methods</a> and normalized for constitutively expressed luciferase activity. The results are expressed as normalized ATF6 luciferase activity relative to untreated (Med.) cells. Values represent mean ± SD from three independent experiments, *<i>P</i><0.05, **<i>P</i><0.01, vs. untreated control, <i>ns</i>: no significant.