Extended Data Fig. 2: Genome-wide CRISPR/Cas9 knockout screening identifies genes increasing the D-Cys toxicity in A549 cells.

a, Volcano plot showing the most significant proteins whose depletion boosts D-Cys toxicity. Among the top hits are several glycolytic genes and the mitochondrial ISC protein glutaredoxin (GLRX5); SLC3A12 and SLC7A11 (red bars) are mentioned for comparison. Q values were calculated using MAGeCK, which includes a correction for multiple comparisons. See also Supplementary Table 2 for detailed results. b, c, Validation of CRISPR/Cas9 KO of SLC3A2 (CD98), SLC7A11 (xCT), or NFE2L2 (NRF2) genes in A549 cells by immunoblotting of corresponding proteins (molecular masses in parentheses). Representative blots of n = 3 (b) and n = 2 (c) biological replicates. d, A549 cells were cultured in full culture medium in the absence (–) or presence of the reductant TCEP (375 μM), in the absence (Ctrl) or presence of the xCT inhibitor erastin (1 μM). Samples either did not receive additional Cys supplementation (w/o), or were supplemented with 500 μM L-Cys or D-Cys for a total of three days as indicated. Cells were harvested and total protein yield was determined (mean ± SD). The efficiency of erastin treatment was analyzed by 2-way repeated measures ANOVA and Bonferroni posttest; symbols indicate matching samples of n = 3 biological replicates. e, Wild-type or knock-out (KO) A549 cells were cultured for three days in full culture medium in the presence of erastin (Era) and/or 500 μM D-Cys as indicated. Intracellular L-Cys and D-Cys levels were determined as a measure of cellular Cys import. Results are mean ± SD of n = 3 biological replicates for cells cultured in the presence (D-Cys) or absence (w/o) of D-cys; n = 2 biological replicates (consisting of 3 technical replicates each) for the other conditions. Unpaired two-tailed Student’s t-tests were used for comparison between cells cultured with and without (w/o) D-Cys. f, BEAS-2B cells were cultured for three days in full culture medium in the absence or presence of 500 μM D-Cys as indicated, and the cellular L- and D-Cys content was determined in n = 2 experimental series as a measure of Cys import.

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