Figure 1 for the manuscript of Zhao et al, 2025 Endothelial NAD+-dependent return to quiescence is required for angiogenesis

Description

These data represent the results of NAD+ turnover in human umbilical vein endothelial cells (HUVECs). Isotope tracers [2,4,5,6-2H] NAM and [U-13C] glucose were used respectively to study the salvage synthesis of NAD+ in proliferating endothelial cells (PECs), contact-inhibited quiescent endothelial cells (QECs) and ECs transitioning from proliferation to quiescence (PtoQ). We found that QECs and PtoQ ECs displayed enhanced NAD+ turnover compared with PECs. In the experiments, cells were grown in 6-cm dishes and incubated with the growth medium containing [2,4,5,6-2H] NAM (Cambridge Isotope Laboratories, DLM-6883, 40 μM) or U-13C6 glucose (Cambridge Isotope Laboratories, CLM-1396, 10 mM) for NAD+ labeling. After incubation, cells were washed with 37°C saline, and metabolites were extracted by adding 200 μl pre-chilled buffer (40% methanol, 40% acetonitrile, 20% water and 0.5% formic acid). Samples were scraped on dry ice, transferred into a centrifuge tube, neutralized with 15% NH4HCO3, and centrifuged at 16,000 g for 10 min at 4°C. The supernatants were transferred to plastic vials for liquid chromatography-mass spectrometry (LC-MS) analysis. Targeted measurements of NAD+ derivatives were achieved on a quadrupole orbitrap mass spectrometer coupled to a Vanquish UHPLC system (Thermo Fisher Scientific). The data was generated using negative ion mode with a scan range of 65-835 m/z and resolution of 140,000, and normalized by the ratio of labeled tracers to total metabolites.

Institutions

• University of Pennsylvania

Categories

Funders

• American Heart Association

  United States

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