Extended Data Fig. 4: Effects of glutathione synthesis inhibition or extraction with the conjugating agent NEM on cysteine fate abundances.
From: Excess cysteine drives conjugate formation and impairs proliferation of NRF2-activated cancer cells
(A) Schematic of cysteine metabolism and glutathione (GSH) synthesis, highlighting metabolic branch points of each. Buthionine sulfoximine (BSO) is an inhibitor of the glutathione synthesis enzyme glutamate-cysteine ligase (GCL). (B) Volcano plot showing differential CYS fate abundances. Each point represents a CYS fate. X-axis denotes the log2 fold change of CYS fates as measured by LC–MS metabolomics between SSP25 cells cultured in 200 μM CYS2 for 24 h with vehicle or 50 μM BSO. Y-axis shows the negative log10-transformed q-value derived from multiple unpaired t tests. Features with at least a two-fold depletion by BSO and an adjusted q-value < 0.05 (FDR correction) are highlighted in blue as ‘GCL-dependent’. Features that do not meet these criteria are highlighted in green as ‘GCL-independent’. n = 3 replicate wells per condition. (C) Volcano plot of differential CYS fate abundances from SSP25 cells with standard extraction (80% MeOH) or NEM extraction (80% MeOH with 2 mM ammonium formate in water, pH 7 and 0.5 mM N-methylmaleimide). 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. Samples with a log2 fold change labelled ‘Inf’, were undetectable in +NEM extraction conditions. Features with a fold change < −1 and adjusted q-value < 0.05 (FDR correction) are highlighted in the lighter shade as ‘Depleted by NEM’. Features that do not meet these criteria are highlighted the darker shade as ‘Resistant to NEM’. n = 3 replicate wells per condition. (D) Ion counts measured by LC–MS metabolomics from the same dataset as in (C) for three NEM-conjugated variants of the thiol-containing parent metabolites cysteine (CYS-NEM, P < 0.0001), N-acetylcysteine (NAC-NEM, P < 0.0001), and glutathione (GSH-NEM, P < 0.0001). Error bars are SEM. n = 3 replicate wells per condition. Statistical significance was assessed using unpaired two-tailed Student’s t-test (D). ****P < 0.0001.
