Trifluoroacetamide

Fatty acid oxidation: ECs were incubated in fully supplemented EBM2 medium with 100 μM unlabeled palmitate and 50 μM carnitine. Cells were incubated for 2 hours in growth medium containing 2 μCi/ml [9,10-³H]-palmitate^{23 ,24} . Thereafter, supernatant was transferred into glass vials sealed with rubber stoppers. ³H₂O was captured in hanging wells containing a Whatman paper soaked with H₂O over a period of 48 hours at 37°C to reach saturation²⁵ . Radioactivity was determined by liquid scintillation counting. Glycolysis: Glycolysis was measured analogously to fatty acid oxidation (cf supra) using 80 mCi/mmol [5-³H]-D-glucose (Perkin Elmer)²³ . ¹⁴C-glucose oxidation: Cells were incubated for 6 hours in growth medium containing 100 μCi/mmol [6-¹⁴C]-D-glucose. Thereafter, 250 μl of 2 M perchloric acid was added to each well to stop cellular metabolism and wells were immediately covered with a 1× hyamine hydroxide-saturated Whatman paper. Overnight absorption of ¹⁴CO₂ released during oxidation of glucose into the paper was performed at room temperature, and radioactivity in the paper was determined by liquid scintillation counting. ¹⁴C-glutamine oxidation: was performed similarly as glucose oxidation, except that we used 0.5 μCi/ml [U-¹⁴C]-glutamine as tracer. Palmitate or hypoxanthine mediated RNA and DNA synthesis: was measured by the incorporation of ¹⁴C into RNA or DNA using 100 μCi/mmol [U-¹⁴C]-palmitate or [8-¹⁴C]-hypoxanthine and was corrected for the total amount of RNA or DNA per sample. Total RNA and DNA were isolated using commercially available column-based DNA and RNA extraction kits (Qiagen) or using Trizol as an alternative extraction method for RNA or DNA. ATP coupled oxygen consumption: Cells were seeded at 40,000 cells per well on Seahorse XF24 tissue culture plates (Seahorse Bioscience Europe, Copenhagen, Denmark). The measurement of oxygen consumption was performed at 10 min intervals (2 min mixing, 2 min recovery, 6 min measuring) for 3 hours using the Seahorse XF24 analyzer. For ATP coupled oxygen consumption, measurements were performed before and after oligomycin (1.2 μM) treatment. Energy balance assessment: 1.5 × 10⁶ cells were harvested in ice cold 0.4 M perchloric acid supplemented with 0.5 mM EDTA. pH was adjusted by adding 100 μl of 2 M K₂CO3. 100 μl of the mixture was injected onto an Agilent 1260 HPLC equipped with a C18-Symmetry column (150 × 4.6 mm; 5 μm) (Waters), thermostated at 22.5 °C. Flow rate was kept constant at 1 ml/min. A linear gradient using solvent A (50 mM NaH₂PO4, 4 mM tetrabutylammonium, adjusted to pH 5.0 using H₂SO4) and solvent B (50 mM NaH₂PO4, 4 mM tetrabutylammonium, 30% CH₃CN, adjusted to pH 5.0 using H₂SO4) was accomplished as follows: 95% A for 2 min, from 2 to 25 min linear increase to 100% B, from 25 to 27 min isocratic at 100% B, from 27 to 29 min linear gradient to 95% A and finally from 29 to 35 min at 95% A. Detection of ATP, ADP and AMP occurred at 259 nm. GSSG/GSH ratio measurement. Samples were collected in 300 μl 5% TCA (trichloro-acetic acid, Sigma). 50 μl was loaded onto an Ultimate 3000 UPLC (Thermo Scientific, Bremen, Germany) equipped with a Acquity UPLC HSS T3 column (cat # 186003976; 2.1 × 5 mm; 1.8 μm particles; Waters) in line connected to a Q Exactive mass spectrometer (Thermo Fisher Scientific). A linear gradient was carried out using solvent A (0.05% formic acid) and solvent B (60% methanol, 0.05% formic acid). Practically, samples were loaded at 99% solvent A and from 10 to 12 min a ramp to 100% solvent B was carried out. From 15 to 16 min the column returned to 99% solvent A and the run was stopped at 21 min. Elution of GSH and GSSG occurred at 3 and 5.5 min respectively (isocratic separation). Flow rate was constant at 250 μl/min and the column temperature was kept constant at 37°C. The mass spectrometer operated in targeted SIM mode following the ions m/z 311.11456 and 308.59499 (GSH and GSSG respectively) using the ion 445.12003 as lock mass. The mass spectrometer ran in positive polarity, the source voltage was 3.0 kV, and the capillary temperature was set at 350°C. Additional sheath gas flow was put at 35 and auxiliary gas flow rate at 10. Auxiliary gas heater temperature was put at 60°C. AGC target was put at 1e5 ions with a maximum ion injection time of 200 ms) acquired at a resolution of 70 000. For the data analyses we manually integrated the peaks representing GSH and GSSG using the Thermo XCalibur Qual Browser software (Thermo Scientific) and data is represented as area of the respective GSH and GSSG peaks. Determination of ¹³C-palmitate, glucose and glutamine incorporation in metabolites and total metabolite levels: For ¹³C-carbon incorporation from palmitate in metabolites, cells were incubated for 48 hours with labeled substrates (confirmation of steady state at that time was confirmed, see Extended Data Fig. 5). For ECs, [U-¹³C]-palmitate labeling was done in two ways: (1) “100% labeling”, whereby all cold palmitate in M199 culture medium (120 μM) was replaced by 120 μM [U-¹³C]-palmitate using M199 medium, containing charcoal stripped serum (which does not contain any fatty acids); and (2) “50/50% labeling”, whereby 100 μM [U-¹³C]-palmitate was added to the EGM2 culture medium containing 100 μM cold palmitate. Both types of labeling yielded similar data and were thus pooled. For comparison with cancer cells, only the 100% labeling strategy was used. Similar labeling methods were used for glucose (5.5 mM) and glutamine (2 mM). Labeling with the algal [U-¹³C] fatty acid mix was performed by using 100% labeling; this fatty acid mix contained 50 μM palmitate. Metabolites for the subsequent mass spectrometry analysis were prepared by quenching the cells in liquid nitrogen followed by a cold two phase methanol-water-chloroform extraction^{7 (link),26} . Phase separation was achieved by centrifugation at 4°C and the methanol-water phase containing polar metabolites was separated and dried using a vacuum concentrator ^{24 ,61}. The dried metabolite samples were stored at −80°C^{7 (link),26} . Polar metabolites were derivatized for 90 min at 37°C with 7.5 μl of 20 mg/ml methoxyamine in pyridine and subsequently for 60 min at 60°C with 15 μl of N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide, with 1 % tert-butyldimethylchlorosilane^{7 (link),26} . Isotopomer distributions and metabolite levels were measured with a 7890A GC system (Agilent Technologies) combined with a 5975C Inert MS system (Agilent Technologies). One microliter of sample was injected onto a DB35MS column in splitless mode using an inlet temperature of 270 °C^{7 (link),26} . The carrier gas was helium with a flow rate of 1 ml min⁻¹. Upon injection, the GC oven was held at 100°C for 3 min and then ramped to 300 °C with a gradient of 2.5 °C min⁻¹. The MS system was operated under electron impact ionization at 70 eV and a mass range of 100–650 amu was scanned. Isotopomer distributions were extracted from the raw ion chromatograms using a custom Matlab M-file, which applies consistent integration bounds and baseline correction to each ion²⁷ . In addition, we corrected for naturally occurring isotopes using the method of Fernandez et al²⁸ . For relative metabolite levels, the total ion count was normalized to the internal standards norvaline and glutarate and to the protein content^{7 (link),26} . To correct for enrichment dilution, we used previously reported methods^{7 (link),29} , i.e. we divided the fractional contribution of a labeled metabolite of interest by the fractional contribution of its precursor (calculated by the formula below).
The total contribution of carbon was calculated using the following equation^{7 (link),29} :
$$\text{Total contribution of carbon}={\Sigma }_{i=0}^{n}i\ast m_i∕(n\ast {\Sigma }_{i=0}^n{m}_i)$$
Herewith, “n” is the number of C atoms in the metabolite, “i” represents the different mass isotopomers and “m” refers to the abundance of a certain mass. Glycolytic carbon contribution was calculated based on [U-¹³C]-glucose labeling and label dilution in pyruvate^{7 (link)}. For total metabolite levels, arbitrary units of the metabolite of interest were normalized to the protein content. A time-course experiment of the incorporation of [U-¹³C]-glucose, [U-¹³C]-glutamine and [U-¹³C]-palmitate in TCA intermediates demonstrated that the incorporation values reached a pseudo-isotopic steady state within experimental measurement precision (Extended Data Fig. 5). determination of dNTP levels by RT-PCR: dNTP levels were determined by using a fluorescence-based PCR assay³⁰ using G1 sorted ECs, identified as CherryRed⁺ Venus⁻ cells upon transduction with a FUCCI construct³¹ . Determination of ¹³C-palmitate or ¹³C-acetate incorporation in UMP and UTP: Cells were labeled with [U-¹³C]-palmitate (100% labeling with 100 μM [U-¹³C]-palmitate; see above) or [U-¹³C]-acetate (20 mM supplementation with [U-¹³C]-acetate) for 48 hours and were then collected in 500 μl ice cold acetonitrile buffer (50% methanol, 30% acetonitrile and 20% water). Samples were spun for 5 min and supernatants were dried down and were then reconstituted in 50 μl of HPLC-grade water, vortexed, centrifuged, and transferred into HPLC vials. LC-MS/MS analysis was done on a Waters Xevo TQ-S mass spectrometry was coupled to an H-Class UPLC system. Metabolites were separated by polarity using Supelco Ascentis Express C18 column (2.7 μm particle size, 5 cm × 2.1 mm). LC parameters are as follows: autosampler temperature, 5 °C; injection volume, 5 μl; column temperature, 50 °C; flow rate over 11 min: t = 0, 0.4 ml/min; t = 2, 0.3 ml/min; t = 3, 0.25 ml/min; t = 5, 0.15 ml/min; t = 9, 0.4 ml/min; t = 11, 0.4 ml/min. The LC solvents were Solvent A: 10 mM tributylamine and 15 mM acetic acid in 97:3 water:methanol (pH 4.95); and Solvent B: methanol. Elution from the column was performed over 11 min with the following gradient: t = 0, 0% B; t = 1, 0% B; t = 2, 20% B; t = 3, 20% B; t = 5, 55% B; t = 8, 95% B; t = 8.5, 95% B, t = 9, 0% B; t = 11, 0% B. Mass spectra were acquired using negative-mode electrospray ionization operating in multiple reaction monitoring (MRM) mode. The capillary voltage was 3000 V, and cone voltage was 50 V. Nitrogen was used as cone gas and desolvation gas, with flow rates of 150 l/h and 600 l/h, respectively. The source temperature was 150 °C, and desolvation temperature was 500 °C. Argon was used as collision gas at a manifold pressure of 4.3 × 10⁻³ mbar. Collision energies and source cone potentials were optimized for each transition using Waters QuanOptimize software. Data were acquired and analyzed using MassLynx 4.1 and QuanLynx software. Isotope labeling data was corrected for the natural abundance of different isotopes using IsoCor³² . determination of rNTP levels by LC-MS: rNTP extracted with the same method as described for UTP and UMP. Additionally, ¹³C-internal standard (generated by based on fully labeled yeast extracts³³ ) were spiked into the extraction solution. rNTP concentrations were determined with the same LC-MS method as described for UTP and UMP. All samples were normalized to the ¹³C-internal standard and protein content.

The following reagents and authentic standard compounds were obtained
from (suppliers): water, isopropanol, and acetonitrile (Fisher Optima); pyridine
(Acros Organics); C8 – C30 fatty acid methyl esters
[FAMEs], methoxyamine hydrochloride [MeOX],
ethoxyamine hydrochloride [EtOX],
N-methyl-N-(trimethylsilyl)-trifluoroacetamide
[MSTFA],
N-methyl-N-(trimethyl-d9-silyl)-trifluoroacetamide
[MSTFA-d9], ammonium formate, formic acid, and
N-methyl-L-alanine (Sigma-Aldrich);
2′-O-methyluridine-5′-triphosphate,
3′-O-methyluridine-5′-triphosphate,
5-methyluridine-5′-triphosphate (TriLink BioTechnologies);
4-hydroxypropofol-1-O-β-D-glucuronide, and
4-hydroxypropofol-4-O-β-D-glucuronide (Toronto
Research Chemicals).
All metabolites extraction procedures are kept on ice, the quantities
for sample aliquots were 25 μL for blood plasma,
5×10⁶ for cells, 5 mg for tissues, 2 mL for algae
cultures. Metabolites were extracted with 1,000 μL degassed
acetonitrile:isopropanol:water (3:3:2, v/v/v), and then homogenized,
centrifuged, decanted, and evaporated. Extracts were cleaned by 500 μL
degassed acetonitrile:water (1:1, v/v) to remove triglycerides and membrane
lipids, and evaporated again. For GC-MS analysis, internal standards C8
– C30 FAMEs were added to determine the retention index. The dried
samples were derivatized with 10 μL MeOX (or EtOX) in pyridine and
subsequently by 90 μL MSTFA (or MSTFA-d9) for trimethylsilylation of
acidic protons. For LC-MS analysis, the extracted samples were resuspended in 50
μL acetonitrile:water (4:1, v/v) and submitted to instrument.