Figure 2
From: Inactivation of NUPR1 promotes cell death by coupling ER-stress responses with necrosis
Nupr1 deficiency induces a decrease in ATP production by OXPHOS suppression. MiaPaCa2 cells were transfected with siCtrl or two different siRNA against Nupr1 for 48 h. Cells were then incubated with thapsigargin, brefeldin A, or tunicamycin at 1 μM for another 24 h period in the presence or not of Z-VAD-FMK (10 μM) or Nec-1 (40 μM), and ATP production was measured (A); data values were normalized to the untreated siCtrl. 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. Mitochondrial respiration reflected by oxygen consumption rate (OCR) levels was measured in MiaPaCa2 cells transfected with siCtrl, siNUPR1-1 or siNUPR1-2 for 72 h (B). Mitochondrial respiration was also measured in 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 (C). Finally, we measured mitochondrial respiration in pancreatic acinar cells from WT (Nupr1+/+) and Nupr1−/−, mice (D). Only statistically significant differences (p values ≤ 0.05) were shown. The OCR was measured under basal conditions or following the addition of oligomycin, FCCP, or rotenone/antimycin A. The rates of OCR for basal respiration, maximal respiration, spare capacity, and ATP production were quantified as described in the Materials and Methods section. The OCR was again determined in MiaPaCa2 cells transfected with siCtrl, siNUPR1-1, or siNUPR1-2 when cells were challenged with UK5099, BPTES, or etomoxir; inhibitors of glucose oxidation, glutaminase, and carnitine palmitoyl-transferase 1 A, respectively (E). Total RNA was extracted to monitor the mRNA levels of the genes involved in the Krebs cycle using RT-qPCR (F). Data are means of triplicates ± SEM. For each treatment, statistically significant differences (*p < 0.05, **p < 0.01, ***p < 0.001) from siCtrl are shown.
