Extended Data Fig. 1: A robust method for 18O2 labelling of human cells.
From: The polar oxy-metabolome reveals the 4-hydroxymandelate CoQ10 synthesis pathway
a, Schematic of 18O2 labelling. A closed system chamber is flushed multiple times with N2 to remove 16O2. A gas mixture containing 18O2 and CO2 is pulsed into the closed chamber to reach the desired oxygen concentration. At the assay endpoint, the chamber is opened, cells are extracted, and metabolites separated and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). b, Oxygen measurements after N2 flush, followed with or without pulses of O2:CO2 gas mixture in the closed chamber containing tissue culture plates and media (n = 3 technical replicates each). c–e, Oxygen percentage of O2-labelling experiments performed at 3% (c), 1% (d), and 0.2% (e) 16O2 or 18O2. (n = 2 technical replicates each). f, Cells were treated with several concentrations of MG132, DFO (an iron chelator), IOX1 (a dioxygenase inhibitor), or in combination at 5% O2 for 24 h. Immunoblots of HIF1α with ERK2 as a loading control. g, Immunoprecipitation of HIF1α to determine its hydroxylation (P564-OH) levels by the indicated inhibitors. Immunoblots of HIF1α and HIF1α P564-OH are shown, with ERK2 serving as a loading control. Experiments were performed once for optimization of drug concentrations (f, g). Graphs represent mean ± s.d. (c–e).
