Fig 2: SFXN4 knockout alters cellular iron metabolism. (A) In-gel aconitase activity of mitochondrial and cytosolic aconitase. (B) Cytosolic aconitase activity in scramble and SFXN4 KO K562 cells. (C) Western blot of SFXN4, TFR1 and FTH from scramble and SFXN4 KO K562 cells. (D) IRE binding activity of IRP1 and IRP2 in scramble and SFXN4 KO K562 cells determined by gel retardation assay. (E) Cytosolic labile iron pool in scramble and SFXN4 KO K562 cells. Shown are means and standard deviations of 4 replicas in a representative experiment of 3 independent experiments. Images are representative of three independent experiments.

Fig 3: Depletion of SFXN4 reduces Fe-S cluster assembly and affects the stability of Fe-S cluster proteins. (A) Relative fluorescence of GRX2-Fe-S dependent sensors in siSFXN4 and siNFS1 transfected HEK293 cells compared to siCon. (B) Mean and standard deviation of fold change in fluorescence intensity from three independent experiments. (C) Immunoblot showing the relative levels of Venus N173 and C155 (sensor plasmids) in HEK293 cells that were transfected with a siRNA targeted to SFXN4, NFS1, and a control siRNA. (D) Immunoblot of indicated Fe-S cluster proteins in SFXN4 KO cells and scramble K562 cells. Images are representative of three independent experiments. Means and standard deviations of quantified data from three independent experiments are shown under the blot. SDHB (succininate dehydrogenase complex iron sulfur subunit B), a component of respiratory complex II; ACO2 (mitochondrial aconitase), a citric acid cycle enzyme; PPAT (phosphoribyosyl pyrophosphate amido transferase), an enzyme that catalyzes the first step of the denovo purine nucleotide biosynthetic pathway; POLD1 (large catalytic subunit of DNA polymerase delta), the large catalytic subunit of the DNA polymerase delta (Polδ) complex.

Fig 4: Dysregulation of the citric acid cycle in rods with chronically active HIFs. a,b Significantly downregulated citric acid cycle associated enzymes in photoreceptors segments (PS) or outer nuclear layer (ONL) of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$rod^{\varDelta \ Vhl}$$\end{document}rodΔVhl mice at 2.5 months of age. Protein levels are shown as boxplots with the median and 25% and 75% percentiles (a) or bar graphs representing normalized abundance ratios of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$rod^{\varDelta \ Vhl}$$\end{document}rodΔVhl/ctrl (b). N = 6 mice per genotype. The samples with the highest (red) and lowest (blue) Cre levels are indicated. For statistical analysis on proteomics data see Methods. nd, not detected. *: p \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\le$$\end{document}≤ 0.05. **: p \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\le$$\end{document}≤ 0.01. c Representation of the citric acid cycle with red arrows marking significantly downregulated proteins in the PRs of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$rod^{\varDelta \ Vhl}$$\end{document}rodΔVhl mice. d-f Representative immunofluorescence labeling for SDH subunit A (SDHA; d), citrate synthase (CS; e), and aconitate hydratase (ACO2; f) in the PS regions of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$rod^{\varDelta \ Vhl}$$\end{document}rodΔVhl, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$rod^{\varDelta \ Cox10}$$\end{document}rodΔCox10 and ctrl mice at time points as indicated (top panels). Pixel intensity (PI) profiles through the PS (bottom panels). White arrows: regions with reduced labeling. Scale bars, 50 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upmu$$\end{document}μm. N = 3 mice per genotype. g Dysregulation of OXPHOS and the TCA cycle in rods with chronic HIF activation is associated with impaired of OS biogenesis and leads to PRs degeneration

Fig 5: Schematic of mechanisms of AGEs–CuCl2-induced cuproptosis via ATF3/SPI1/SLC31A1 pathway in diabetic cardiomyopathy. Excessive AGEs and copper in diabetes upregulated copper importer SLC31A1 through ATF3/SPI1, thereby mediating copper accumulation in cardiomyocytes, disturbing copper homeostasis and promoting cuproptosis. This promoted the decline of Fe–S cluster protein (FDX1, LIAS, NDUFS8 and ACO2) and decreased lipoylation of DLAT- and DLST-aggravated mitochondrial dysfunction in cardiomyocytes and resulted in myocardial dysfunction.