Extended Data Fig. 3: Characterization of glycolysis metabolites and cysteine fates that incorporate carbons from glucose.

(A) Relative abundances of glucose fates and CYS fates measured by LC–MS metabolomics from SSP25 cells cultured in standard DMEM media (25 mM glucose) or in low glucose DMEM (250 μM glucose) for 6 h. Relative abundance was calculated to average ion count of standard media group for each metabolite. n = 3 replicate wells per condition. Glucose, Glucose-6-P, Ribose-5-P, Glyceraldehyde-3-P, DHAP, Lactate P < 0.0001. PEP P = 0.8290. Pyruvate P = 0.8974. 2SC P = 0.1109. GSH P = 0.6809. Lac-GS P = 0.1586. GSF = 0.9795. GSSG P = 0.0058. (B) Volcano plots of differential CYS fate abundances from NRF2^on cell lines (SSP25, OCUG1, and TFK1). Each point represents a CYS fate. X-axis denotes the log₂ fold change of CYS fates as measured by LC–MS metabolomics between cells cultured in standard DMEM media (25 mM glucose) or in low glucose DMEM (250 μM glucose) for 6 h. Y-axis shows the negative log₁₀-transformed q-value derived from multiple unpaired t tests. Features with a log₂ fold change of < -1 and adjusted q-value < 0.05 (FDR correction) are highlighted and listed below each graph. n = 3 replicate wells per condition. (C) Volcano plot of differential CYS fate abundances from SSP25 cells cultured in vehicle or 5 mM 2-deoxyglucose (2DG) for 6 h. Each point represents a CYS fate. X-axis denotes the log₂ fold change of CYS fates as measured by LC–MS metabolomics between cells Y-axis shows the negative log₁₀-transformed q-value derived from multiple unpaired t tests. Features with a fold change < -1 and adjusted q-value < 0.05 (FDR correction) are highlighted and listed below the graph. n = 3 replicate wells per condition. (D) Summarized table of unknown CYS fates depleted in low glucose or upon treatment with 2DG. (E) Fractional isotopologue distribution of glycolytic intermediates glucose-6-phosphate (G6P), dihydroxyacetone phosphate, phosphoenolpyruvate, and pyruvate as measured by LC–MS metabolomics after culturing SSP25 cells in media containing [U-¹³C] glucose for the indicated times. n = 3 replicate wells per condition. (F) Fractional isotopologue distribution of 10 unknown CYS fates with sugar-like added masses as measured by LC–MS metabolomics after culturing SSP25 cells in media containing [U-¹³C] glucose for the indicated times. n = 3 replicate wells per condition. (G) Fractional isotopologue distribution of lactoylglutathione (Lac-GS) as measured by LC–MS metabolomics after culturing SSP25 cells in media containing [U-¹³C] glucose for the indicated times. n = 3 replicate wells per condition. (H) Fractional isotopologue distribution of CYS conjugate C270_5.1, a fate not predicted to derive carbon from glucose, as measured by LC–MS metabolomics after culturing SSP25 cells in media containing [U-¹³C] glucose for the indicated times. n = 3 replicate wells per condition. Error bars are SEM. n = 3. Statistical significance was assessed using two-way ANOVA with Sidak’s correction for multiple comparisons (A). ns = not significant, **P < 0.01, ****P < 0.0001.

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