Tracefinder software version 4

All data except the untargeted metabolomics analysis were quantified by integrating the area under the curve of each metabolite using MassHunter Quant (Agilent Technologies). Pathway enrichment analyses were performed using Metaboanalyst software (https://www.metaboanalyst.ca/) based on KEGG metabolic pathways. Pathway analysis was performed comparing the relative metabolite levels between BAD SAHB_A SD and vehicle, in the presence of cytokines (Figure S1A). The impact score represents the number of nodes contained in the data and the adjusted p value was obtained using Holm adjustments (Xia and Wishart, 2011 (link)). Targeted processing of GSH and isotopolog for data collected using LCMS “method D” was conducted using TraceFinder software version 4.1 (Thermo Fisher Scientific). Compound identities were confirmed using reference standards. GSH metabolite isotopolog abundances were normalized to the recovery of internal standards inosine-¹⁵N₄, thymine-D₄ and glycocholate-D₄. Total ion counts of each mass isotopolog of GSH were corrected for natural abundances of ¹³C and fractional labeling out of the total pools of GSH were calculated in Excel.

After treating cells with CKi or MitoCKi, cells were collected and washed with PBS. 250 μL of 80% MeOH containing 0.05 ng/μL thymine-D4, 0.05 ng/μL ¹⁵N4-inosine, and 0.10 ng/μL glycocholate-D4 was added to the cell pellet and vortexed. Cell debris was removed by centrifugation for 10 mins, 15,000 × g, 4 °C. The supernatant was transferred into a fresh tube and centrifuged again. The supernatant was collected and subjected to LC-MS analysis as described previously³¹ . Briefly, metabolite extracts were loaded onto a Luna-HILIC column (Phenomenex) with 10% mobile phase A (20 mM ammonium acetate and 20 mM ammonium hydroxide in water) and 90% mobile phase B (10 mM ammonium hydroxide in 75:25 v/v acetonitrile/methanol). Analysis was carried out using a QExactive HF-X mass spectrometer (Thermo Fisher Scientific). Negative and positive ion modes were used with full scan analysis over m/z 70–750 m/z at 60,000 resolution, 1e6 AGC, and 100 ms maximum ion accumulation time. Targeted processing of a subset of known metabolites was conducted using TraceFinder software version 4.1 (Thermo Fisher Scientific). Compound identities were confirmed using reference standards. In all cases metabolite abundance was normalized using internal standards and relative changes were assessed by comparison with metabolite extracted from the same sample type.