Extended Data Fig. 9: D-Cys induces Fe-S protein defects particularly at low cell densities.
From: d-cysteine impairs tumour growth by inhibiting cysteine desulfurase NFS1
a, A549 cells were seeded at low density (LD, 5×103 cells/cm2) or high density (HD, 60×103 cells/cm2), cultured in the absence (w/o) or presence of 500 μM D-Cys for LD cultures and 1 mM for HD cultures. After three days cells were harvested and counted; experiments were done in technical triplicates and performed twice; 2-way ANOVA followed by Sidak’s multiple comparisons test; **** P < 0.0001. b, c, Indicated numbers of A549 cells were seeded into 25 cm2 flasks, cultured in the absence (w/o) or presence of 500 μM D-Cys for a total of three days and harvested. Protein yield of each culture at harvest (b), and relative protein yield of D-Cys-treated cultures in comparison to untreated cultures (c) are presented. d, A549 cells were cultured at LD or HD conditions as indicated in (a). After 15 h, the cellular D-Cys content was measured by the D-Cys luciferase assay, and expressed relative to the HD value. e, Protein levels of xCT and CD98 in cells from (b) were determined by immunoblotting. α-tubulin (TUBA) served as loading control. f, Immunofluorescence confocal analysis of xCT and phalloidin in A549 cells cultured at LD (top) to HD (bottom) conditions (left panels). Note the colocalization of xCT and phalloidin at HD. Z sections taken at the horizontal middle position of the respective XY view (left) are shown in right panels. Images are representative of n = 2 biological replicates. g, h, Cell samples from (b) were analysed by immunoblotting against the indicated mitochondrial proteins or lipoyl cofactor. VDAC1 served as loading control. C-I to C-V, OXPHOS complexes I to V. i, j, Total cell lysates of cells from (c) were analysed for the specific enzyme activities of total cellular aconitase (ACO) and succinate dehydrogenase (SDH, respiratory complex II). k, Cell samples were analysed by immunoblotting against the indicated cytosolic-nuclear CIA and Fe-S proteins. TUBA served as loading control. l, Crude membrane preparations of cells from (b) were analysed for the specific enzyme activity of cytochrome c oxidase (COX, respiratory complex IV). Representative blots and images are shown (e–h, k). Numbers in parentheses indicate observed molecular masses. Data obtained from n = 3 biological replicates are presented as mean ± SD, with individual symbols indicating matching samples (b–d, i, j, l); Comparisons were performed by 2-way repeated measures ANOVA and Bonferroni posttests (b, c, i, j, l); * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.
