Extended Data Fig. 6: D-Cys induces Fe-S protein defects in xCT/CD98-overproducing HeLa cells.

HeLa cells were transiently transfected to co-express C-terminally FLAG-tagged CD98 and xCT, or the reference protein EGFP as indicated. Cells were cultured in the absence (w/o) or presence of 500 μM D-Cys for three days (cf. Extended Data Fig. 3d, e). a, b, Cell extracts were analysed by immunoblotting of the indicated mitochondrial proteins or the lipoyl cofactor. ATP5F1A/B and VDAC1 served as loading controls. c–f, Mitochondria-containing organellar fractions, obtained by digitonin-based cell separation, were analysed for the specific enzyme activities of mitochondrial aconitase (mtAco), succinate dehydrogenase (SDH, respiratory complex II), citrate synthase (CS), and cytochrome c oxidase (COX, respiratory complex IV). g, Cell samples were analysed by immunoblotting of the indicated cytosolic nuclear proteins. α-tubulin (TUBA) served as loading control. h-i, Cytosolic fractions obtained by digitonin-based cell separation were analysed for the specific enzyme activities of cαytosolic aconitase (cytAco) and lactate dehydrogenase (LDH). Representative blots of n = 4 biological replicates are shown. Numbers in parentheses indicate observed molecular masses. C-I to C-V, OXPHOS complexes I to V. Data are presented as mean ± SD; *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. Symbols indicate matching samples of n = 4 biological replicates.

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