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. Author manuscript; available in PMC: 2024 Jun 13.
Published in final edited form as: Science. 2023 Nov 2;382(6672):820–828. doi: 10.1126/science.adf4154

Fig. 3. CRISPR screen identifies [2Fe-2S] cluster assembly as essential for the regulation of SLC25A39 stability.

Fig. 3.

(A) Schematic of the CRISPR screen workflow with mitochondrial sgRNA library for SLC25A39 stability under GSH depletion. (B) (Top) Dot plot showing the distribution of differential CRISPR gene score calculated as (median guide enrichment in the SLC25A39-hi fraction) – (median guide enrichment in the SLC25A39-lo fraction). Pink dots indicate genes in the [2Fe-2S] cluster assembly pathway. The green dot indicates the putative mitochondrial [2Fe-2S] cluster exporter ABCB7. (Bottom) Gene Ontology enrichment analysis of genes with a differential CRISPR gene score lower than −1. (C) Schematic of the mitochondrial Fe-S cluster assembly pathways that highlights the genes that scored (pink and green) in the CRISPR screen. (D) Immunoblots of the indicated proteins in HEK293T cells that express 3xFLAG-SLC25A39. (Top) Cells were transduced with lentivirus expressing Cas9 and control sgRNA or sgRNAs that target iron-sulfur cluster assembly factor HSCB. Cells were treated for 24 hours with BSO (1 mM) and erastin (5 μM) or DMSO as the control. (Bottom) Cells were transduced with lentivirus expressing control sgRNA or sgRNAs targeting ABCB7 (left), or shRNAs targeting GFP or ABCB7 (right). (E) Scatter plot showing log2 fold change and −log10(P value) of proteomics analysis from immunopurified mitochondria of the indicated samples. The x axis represents log2 protein fold change (FC) in isolated mitochondria from HEK293T cells that express shRNA targeting GFP or cysteine desulfurase NFS1 after 24-hour treatment with BSO (1 mM) and erastin (5 μM). The y axis represents log2 fold change (FC) in mitochondrial protein abundance after treating cells with BSO (1 mM) and Erastin (5 μM) versus DMSO as the control. The color grid indicates −log10(P values) and green circles represent [2Fe-2S] cluster–containing proteins. (F) Immunoblots of the indicated proteins from the whole-cell lysates or FLAG-immunoprecipitation from HEK293T cells stably expressing cDNAs for vector or 3xFLAG-SLC25A39, are infected with lentivirus-expressing control sgRNA or sgRNAs for HSCB, and are transiently transfected with AFG3L2(E408Q)-HA cDNA. Cells were treated for 24 hours with BSO (1 mM) and erastin (5 μM) or DMSO as the control.