Fig. 4: Free cysteine defines the proliferation defects caused by excess cysteine stress.
From: Excess cysteine drives conjugate formation and impairs proliferation of NRF2-activated cancer cells
a, Change in cell proliferation rate of SNU308 cells cultured in medium containing either 200 or 800 μM CYS2 upon co-treatment with 50 μM BSO. Each value represents the average result across technical replicates from n = 8 independent experiments, P = 0.0003. b, Relative abundance of intracellular cysteine, measured as CYS–NEM, from SNU308 cells cultured in medium containing either 200 or 800 μM CYS2 treated with either vehicle (DMSO) or 50 μM BSO for 24 h then extracted with NEM and measured by LC–MS metabolomics. Abundances are relative ion counts to the 200 μM CYS2 vehicle group. n = 3 replicate wells per condition, 200 μM CYS2 P = 0.0170, 800 μM CYS2 P < 0.0001. c, Relative abundances of intracellular sugar–CYS conjugates as measured by LC–MS metabolomics from SNU308 cells cultured in medium containing either 200 or 800 μM CYS2 treated with either vehicle (DMSO) or 50 μM BSO for 24 h. Abundances are relative ion counts to cells cultured in 200 μM CYS2 with vehicle for each metabolite. n = 3 replicate wells per condition. 3GC: 200 μM CYS2 P = 0.3876, 800 μM CYS2 P = 0.0032. 1DC: 200 μM CYS2 P = 0.6299, 800 μM CYS2 P = 0.0020. d, Schematic depicting hypothesized model in which pyruvate treatment can impact the availability of free CYS through sequestration of CYS into 2MTDC. e, Cell proliferation rates of SNU308 cells cultured in different medium concentrations of CYS2 with or without 1 mM pyruvate, with or without 0.5 μM erastin. n = 3 replicate wells per condition. 50 μM CYS2 P > 0.9999, 200 μM CYS2 P = 0.1011, 800 μM CYS2 P = 0.0021, 800 μM CYS2 + 0.5 μM erastin CYS2 P = 0.7829. f,g, Relative abundances of 2MTDC (f) or sugar–CYS conjugates (g) as measured by LC–MS metabolomics in SNU308 cells cultured in either 200 or 800 μM CYS2 with or without 1 mM pyruvate. n = 3 replicate wells per condition. 2MTDC: 200 μM CYS2 P = 0.0367, 800 μM CYS2 P = 0.0059. 3GC: 200 μM CYS2 P = 0.0167, 800 μM CYS2 P < 0.0001. 1DC: 200 μM CYS2 P = 0.0523, 800 μM CYS2 P < 0.0001. h, Relative abundance of total intracellular cysteine, measured as CYS–NEM, from SNU308 cells cultured in medium containing either 200 or 800 μM CYS2 with or without 1 mM pyruvate for 24 h, then extracted with NEM and measured by LC–MS metabolomics. Abundances are relative ion counts to the 200 μM CYS2 vehicle group. n = 3 replicate wells per condition. i, Schematic depicting a model where sugar–CYS conjugate abundance may proportionately reflect the free CYS pool, while NEM extraction reveals the total available CYS pool, incorporating both the free CYS and the CYS that is sequestered in reversibly bound metabolites (such as 2MTDC). Error bars show s.e.m. Statistical significance was assessed by unpaired two-tailed Student’s t-test (a) or two-way ANOVA with Sidak’s correction for multiple comparisons (b,c,e–h). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
