Extended Data Fig. 2: Metabolite consumption rates and measurements of intracellular CYS fates identified by RMA tracing in bile duct cancer cell lines.
From: Excess cysteine drives conjugate formation and impairs proliferation of NRF2-activated cancer cells
(A) Schematic depicting quantification of media metabolites and cell biomass over time to calculate metabolite fluxes. (B) Heatmap of the fold change in consumption rates of metabolites for each cell line, relative to the average value of the three NRF2off cell lines. (C) Average media fluxes of individual metabolites, comparing rates between NRF2on cells and NRF2off cells. Left graph consists of amino acids supplied in DMEM, centre graph consists of amino acids not supplied in DMEM, and right graph consists of sugars. Individual fluxes were determined over multiple time points for each cell line, with n = 3 for each time point. Grouped rates shown here are the average of consumption rates across the cell lines of each category, n = 5 for NRF2on and = 3 for NRF2off. Glutamine: P > 0.9999. Cystine: P < 0.0001. Leucine: P > 0.9999. Serine: P > 0.9999. Isoleucine: P > 0.9999. Valine: P > 0.9999. Glycine: P > 0.9999. Lysine: P = 0.9997. Arginine: P > 0.9999. Threonine: P > 0.9999. Phenylalanine: P > 0.9999. Tyrosine: P > 0.9999. Methionine: P > 0.9999. Tryptophan: P > 0.9999. Glutamate: P < 0.0001. Alanine: P = 0.8712. Proline: P > 0.9999. Aspartate: P > 0.9999. Asparagine: P > 0.9999. Glucose: P = 0.9903. Lactate: P = 0.708. (D) Absolute quantification of intracellular cysteine, measured as CYS–NEM, or intracellular glutathione, measured as GSH-NEM, in five bile duct cancer cell lines cultured in 200 μM CYS2 for 2 h and extracted with NEM and measured by LC–MS. n = 3. CYS-NEM: CCLP1 vs SNU308 P < 0.0001, CCLP1 vs TFK1 P < 0.0001, YSCCC vs SNU308 P < 0.0001, YSCCC vs TFK1 P < 0.0001, CCLP1 vs SSP25 P = 0.0064, YSCCC vs SSP25 P = 0.0135. GSH-NEM: CCLP1 vs SNU308 P < 0.0001, CCLP1 vs TFK1 P < 0.0001, YSCCC vs SNU308 P < 0.0001, YSCCC vs TFK1 P < 0.0001, CCLP1 vs SSP25 P = 0.0099, YSCCC vs SSP25 P = 0.0483. (E) Relative media flux of cystine in SNU308 cells cultured in 200 μM CYS2 with vehicle or 200 μM BSO, relative to the cystine flux of untreated cells for 24 h. Negative value indicates net consumption. n = 3. (F) Relative metabolite levels across cell lines for each of nine known CYS fates identified by RMA tracing that had been verified by chemical standards (n = 3). Relative ion counts are calculated as relative to the average of NRF2off cell average for each metabolite. CYS: cysteine, NAC: N-acetylcysteine, GSSG: glutathione disulfide, 2SC: S-(2-succinyl)-cysteine, γ-EC: γ-glutamylcysteine, Lac-GS: lactoylglutathione, GSH: glutathione, GSF: succinated glutathione (also known as succinicGSH or S(1,2-dicarboxyethyl)glutathione), CSA: cysteine sulfinic acid. Numbers in x-axis labels represent the rank of most enriched CYS metabolites in NRF2on cell lines compared to NRF2off cell lines from RMA tracing, corresponding to the row number in Fig. 1f. n = 3. (G) Chromatographic retention times of RMA tracing identified 29 CYS fates, annotated with retention times of known CYS fates, related to Fig. 1g. Error bars are SEM. Statistical significance was assessed using two-way ANOVA with Sidak’s correction for multiple comparisons (C, D) or by unpaired two-tailed Student’s t-test (E). ns = not significant, *P < 0.05, **P < 0.01, ****P < 0.0001. Panel a created in BioRender; Sullivan, L. https://biorender.com/wtofmpq (2026).
