Fig. 3: d-Cys induces severe cellular Fe–S protein defects.
From: d-cysteine impairs tumour growth by inhibiting cysteine desulfurase NFS1
a–k, BEAS-2B and A549 cells were cultured in the absence (w/o) or presence of 500 µM d-Cys for a total of 3 days and analysed. a–c, Cell samples were immunoblotted against xCT and CD98 subunits, the indicated mitochondrial proteins or the lipoyl cofactor. TUBA, VDAC1 and ATP5F1A/ATP5F1B served as loading controls. d–f, Mitochondria-containing organellar fractions obtained by digitonin-based cell separation were analysed for the specific enzyme activities of mitochondrial aconitase (mtAco) (d), succinate dehydrogenase (SDH, respiratory complex II) (e) and cytochrome c oxidase (COX, respiratory complex IV) (f). Comparison by two-way repeated-measures ANOVA and Bonferroni post-test; symbols indicate matching samples of n = 3 biological replicates. g, Electron microscopy of mitochondria from A549 cells cultured for 3 days in the absence (w/o) or presence of 500 µM d-Cys. h, Cell samples were analysed by immunoblotting against the indicated cytosolic and nuclear proteins. Alpha-tubulin (TUBA) served as the loading control. i,j, Immunofluorescence analysis of 53BP1 and γ-H2AX to assess DNA damage in A549 cells cultured in the absence (w/o) or presence of d-Cys (i). The percentage of 53BP1-positive or γ-H2AX-positive cells was determined (j), n = 2 biological replicates. k, Cytosolic fractions obtained by digitonin-based cell separation were analysed for the specific enzyme activity of cytAco (IRP1); comparison by two-way repeated-measures ANOVA and Bonferroni post-test; symbols indicate matching samples of n = 3 biological replicates. Representative blots and images of n = 3 biological replicates are shown. Observed molecular masses in immunoblots are indicated in parentheses. C-I to C-V, OXPHOS complexes I–V. All data are presented as the mean ± s.d.; *P < 0.05; ***P < 0.001; ****P < 0.0001; NS, not significant.
