Long-term NOD treatment results in hypometabolism and thermotolerance.

<p>Three different primary HUVEC isolations were cultured from P2 until P6 in the absence or presence of the indicated NOD concentrations. (a) BiP mRNA expression for cells cultured in the presence of 0.1 and 1 µM NOD was assessed by qPCR. (b) CHOP, BiP, PDI4, and ERO1L mRNA expression for cells cultured in the presence of 0.1 µM NOD was assessed by qPCR. The results in a and b are expressed as normalized mean mRNA levels relative to untreated controls (0, Med) ± SD of 3 replicates for each condition and for all 3 HUVEC isolations. ** <i>P</i><0.01, *<i>P</i><0.05 vs. untreated cells. (c) Intracellular ATP concentration was assessed in HUVEC at P6 upon long-term culture in the absence (medium) or presence of NOD (1 µM). The results are expressed as mean ATP concentration (pmol/5*105 cells) ± SD of 3 replicates for each condition and for all 3 HUVEC isolations. **<i>P</i><0.01 vs. untreated cells (d,e). In separate experiments NOD (1 µM) containing medium was exchanged for normal cell culture medium at P6 and the cells were cultured for 2 (2d) or 5 (5d) days in the absence of NOD. Cells that were continuously cultured in normal cell culture medium (Med.) were used as control. In (d) protein extracts were made and assessed for phosphorylation of AMPK (p-AMPK) by Western blotting. The blots were stripped and re-probed with antibodies directed against β-actin to test for equal loading. The results of a representative blot are depicted, a total of 3 independent experiments were performed. In (e) the cells were subjected to cold storage (4°C) for 24 hrs. Hereafter LDH release was assessed in the supernatants. Each condition was tested in triplicate in 2 independent experiments. The results are expressed as mean LDH release (OD490) ± SD, **<i>P</i><0.01 vs. (Med.) control.