Repression of Fe-S cluster biogenesis factors amplifies NO-induced damage of Fe-S proteins. Exposure of RAW264.7 cells to LPS and IFN-γ led to rapid activations of the Akt, mTOR, HIF-1α, and NF-κB signaling pathways (A) and lactate production (B), followed by an induction of iNOS (A) and a concomitant decrease in the levels of NFS1, ISCU, SDHB, and FECH, aconitase activity, and lipoyl-PDH E2 (C). (D) HeLa cells transfected with NT siRNA or NFS1 siRNA were treated with 400 μM DEA-NO for 1 hour and then washed and incubated in fresh medium for up to 6 hours. In-gel assays indicated that aconitase activity recovered poorly in NFS1-depleted cells compared with control cells. (E) RCII activity and levels of SDHB, FECH, lipoyl-PDH, and lipoyl-KGDH were largely rescued by cotransfection of RAW264.7 cells with pCMV-NFS1-F/M, pCMV-ISCU-F/M, and pCMV-HSC20. In contrast, substitution of ISCU with a construct that expressed an inactive ISCU mutant (ISCU C138S) failed to restore RCII activity and the levels of SDHB, FECH, lipoyl-PDH, and lipoyl-KGDH. **P < .004.