Reduced expression of Fe-S cluster biogenesis factors impairs the regeneration/repair of Fe-S proteins involved in mitochondrial oxidative metabolism. (A) Levels of lipoyl-PDH E2, SDHB, NDUFS1, and FECH in A549 cells decreased in response to silencing of NFS1 and ISCU. (B) Levels of SDHB and NDUFS1 decreased in CRISPR/Cas9 HSC20-knockout HEK293 cell mitochondrial lysates (crRNA HSC20). A scramble CRISPR RNA was used as control (crRNA NT CTRL). (C) Levels of lipoyl-PDH E2, lipoyl-αKGDH E2, SDHB, and NDUFS1 decreased in response to silencing of FXN. (D) Exposure of RAW264.7 cells to LPS and IFN-γ led to a decrease in RCI and RCII activity. (E) Activation of RAW264.7 cells with LPS or LPS/IFN-γ led to decreased mRNA levels of the Fe-S cluster biogenesis factors Nfs1, Iscu, Hscb, Fxn, and Lyrm4 (Isd11). (F) Protein levels of NFS1, ISCU, HSC20, ISD11, SDHB, and FECH and lipoylation of PDH E2 and αKGDH E2 decreased in RAW264.7 cells treated with LPS and IFN-γ. (G) Transcript levels of Iscu, Hsc20, Nfs1, Glrx5, and Lyrm4 decreased in peritoneal macrophages stimulated ex vivo with LPS and IFN-γ. (H) Protein levels of ISCU, NFS1, and HSC20 decreased in peritoneal macrophages stimulated ex vivo with LPS and IFN-γ. Actin or VDAC1 were used as loading controls. All data presented are representative of ≥3 independent experiments. NT, nontargeting.