Fig 4. Deglutathionylation agent, DTT, restores ROS release from skeletal muscle mitochondria treated with disulfiram.

(A) Auto-stimulation of O₂^●-/H₂O₂ release by DTT can be prevented by washing mitochondria. For experiments, mitochondria were not supplemented with substrate. Mitochondria were treated with DTT, incubated for 10 min, and then washed 0–3 times with basic medium. Spontaneous O₂^●-/H₂O₂ release was then recorded. (B) Mitochondria were treated with disulfiram (500 μM) for 20 min, washed once, and then treated with or without DTT (2 mM) for an additional 10 min. Mitochondria were then washed thrice and O₂^●-/H₂O₂ release was tracked using Amplex UltraRed. The final concentration of pyruvate, succinate, or palmitoyl-carnitine was 50 μM. For experiments with pyruvate, malate was also included in the reaction mixture at a final concentration of 50 μM. In palmitoyl-carnitine experiments, mitochondria were pre-incubated in basic medium containing 2 mM carnitine. n = 4, mean±SEM.