Glutamine

NRCMs were incubated in 6-well plates for 5 min or 4–18 h in DMEM media (US Biological; Swampscott, MA, USA) containing 1 mM pyruvate, 4 mM glutamine, and 25 mM [¹³C₆]-glucose (99% purity, microbiological and pyrogen tested; Cambridge Isotope Laboratories, Inc., Tewksbury, MA). Additionally, for compound/inhibitor experiments, FCCP, KA, Oligo, Rot, or 2DG were added to this [¹³C₆]-glucose containing media. After the specified isotope labeling time point, cell reactions were quenched in cold acetonitrile, and extracted in acetonitrile:water:chloroform (v/v/v, 2:1.5:1), as described previously^{31 (link),32 (link),60 (link),61 (link)}, to obtain the polar, nonpolar, and insoluble proteinaceous fractions. The nonpolar (lipid) layer was collected, dried under a stream of nitrogen gas, and reconstituted in 0.1 ml of chloroform:methanol:butylated hydroxytoluene (2:1 + 1 mM) mixture and stored at −80 °C for future analysis. The polar fraction was lyophilized using a Freezone 2.5 L −84 °C benchtop freeze dryer (Labconco, Kansas City, CO, USA). The dried sample was reconstituted in 100 μl 20% acetonitrile and used for LC-MS analysis.

To study whether doxycycline alters metabolic flexibility and mitochondrial metabolism [34 (link),49 (link)], we performed ¹³C-fluxomics as previously [53 (link)]. In short, the incubation was performed for 2 h in 500.000 cells/well, in DMEM without glucose, pyruvate, glutamine and phenol red (Life technologies, Bleiswijk, The Netherlands) supplemented with 50 μM oleic acid in 0.2% BSA, 1 mM glutamine, 50 μM carnitine, and 5 mM [¹³C₆]-glucose (Cambridge Isotope Laboratories, Tewksbury, MA, USA). Every condition was performed in duplicate.
Cells were washed in saline twice, collected in ice-cold methanol-water, chloroform (1:1:2 v/v) and centrifuged at 10,000× g for 10 min. The aqueous phase of the extractions was collected and evaporated. The metabolite pellet was dissolved in 100 µL methanol-water (3:2 v/v) and analyzed by ultra-high-pressure liquid chromatography (Thermo Scientific, Waltman, MA, USA) with a SeQuant ZIC-cHILIC column (PEEK 100 × 2.1 mm, 3.0 µm particle size, Merck, Darmstadt, Germany) at 15 °C coupled to a Thermo Q Exactive (Plus) Orbitrap mass spectrometer (Thermo Scientific, Waltman, MA, USA). Data were acquired in negative-scan mode. Data analysis was performed in Xcalibur software (Thermo Scientific, Waltman, MA, USA). [¹³C]-label enrichment was calculated and corrected based on mass distribution isotopomer analysis [54 (link)].