13c6 glucose

Cells were seeded in 3D conditions using 11 mM ¹³C₆-Glucose (Cambridge Isotope Laboratories) dissolved in RPMI (XXX, ThermoFisher) + 10% dialyzed FBS. Spheroids grew for 5 days and then quenching and metabolite extraction were performed as described above. Metabolite abundances and ¹³C labeling patterns were analyzed by either gas chromatography–mass spectrometry. Mass distribution vectors were extracted from the raw ion chromatograms using Matlab, corrected for naturally occurring isotopes using the method of Fernandez et al ^{72 (link)}, and fractional contribution of carbon was calculated as described by Buescher et al ^{73 (link)}. Palmitate uptake was calculated using the labeled substrate ¹³C16-Palmitic acid and measuring the difference in labeled palmitate in media after 5 days. Palmitate normalized total ion counts from media were corrected by total protein content of each analyzed well. Fractional de novo synthesis of fatty acids was calculated using Isotopomer Spectral Analysis (ISA) during the exposure to the labeled substrate ¹³C₆-Glucose for 5 days.

All solvents and reagents used for metabolite extraction and analysis by LC/MS and DESI imaging were of LC/MS grade. Acetonitrile, methanol and water were purchased from Honeywell (Charlotte, NC, USA), and ammonium bicarbonate, methylenediphosphonic (medronic) acid, ammonium hydroxide and formic acid were purchased from Sigma-Aldrich. The sodium carboxymethylcellulose (CMC) used for preparing zebrafish for DESI imaging was purchased from Sigma-Aldrich. Hydroxychloroquine sulfate was pharmaceutical grade and purchased from Sigma-Aldrich, ¹³C₆-glucose was purchased from Cambridge Isotopes (Tewksbury, MA, USA), and penicillin-streptomycin was purchased from Life Technologies (Carlsbad, CA, USA). The labeled HCQ-D₄ internal standard used for absolute quantitation was purchased from Cayman Chemical (Ann Arbor, MI, USA).

¹³C₆-glucose was purchased from Cambridge Isotope Laboratories and d-fructose-¹³C₆ (587621) was purchased from Sigma-Aldrich. SITA with ¹³C-glucose and ¹³C-fructose allowed for the identification of isotopomer distribution of metabolites. Both ¹³C fructose and glucose were added in glucose-free Dulbecco’s modified Eagle’s medium supplemented with 10% dialysed FBS and 50 µM β-mercaptoethanol in the presence of LPS (10 ng/mL). Metabolites were extracted after 24 h of LPS activation and analysed by liquid chromatography-mass spectrometry (LC-MS) using methods previously described^{48 (link)}. In brief, 1 × 10⁶ BMDMs were washed with cold PBS and metabolites were extracted with 200 µL of ice-cold extraction buffer (methanol, acetonitrile and water (50:30:20)). Samples were centrifuged at 21.1 × g for 10 min at 4 °C and the supernatant was collected for LC-MS analysis. Extracellular metabolites were extracted using 10 µL of culture media added to 490 μL of ice-cold extraction buffer and centrifuged at the aforementioned speed. Supernatants were collected and run through LC-MS analysis. LC-MS machine information and operation is further described in Labuschagne et al.^{49 (link)}. Spectra analysis was performed using the Thermo TraceFinder software.

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.

High-performance liquid chromatography-grade acetonitrile, ethanol, methanol, water, trifluoroacetic acid (TFA), bleomycin (B5507, Lot#SLCG3757), and recombinant isoamylase were purchased from Sigma-Aldrich. α-cyano-4-hydroxycinnamic acid (CHCA) matrix was purchased from Cayman Chemical. Histological-grade xylenes were purchased from Spectrum Chemical. Citraconic anhydride for antigen retrieval was obtained from Thermo Fisher Scientific. Recombinant PNGaseF Prime was obtained from N-Zyme Scientifics (Doylestown, PA, USA). Normal lung fibroblast isolated from adult lung tissue and diseased lung fibroblast isolated from adult idiopathic pulmonary fibrosis lung tissue were purchased from Lonza, USA (Cat# CC-2512 and CC-7231, respectively). ¹³C₆-glucose was purchased from Cambridge Isotope Laboratories, Inc (Cat# CLM-1396-pk).