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. Author manuscript; available in PMC: 2011 Feb 1.
Published in final edited form as: Biochim Biophys Acta. 2009 Nov 24;1797(2):285–295. doi: 10.1016/j.bbabio.2009.11.005

Fig. 7. De-thiolation promotes bioenergetic recovery and prevents cell death.

Fig. 7

Protein-glutathione adducts, bioenergetics, and cell viability in diamide-treated cells exposed to the reducing agent, dithiothreitol. (A) Dithiothreitol (DTT) reverses protein glutathiolation induced by diamide: Cells were treated without or with diamide for 40 min. DTT was then added to the cells to a final concentration of 1 mM for 10 min. The medium was then removed, and the cells were lysed in buffer containing N-ethylmaleimide. Protein-glutathione (PSSG) adducts were assessed by chemiluminescent Western blotting with anti-PSSG antibodies. (B) Group data from panel A. n = 3 per group; *p<0.05 vs. cells not treated with diamide; #p<0.05 vs. diamide-treated cells not exposed to DTT. (C and D) Bioenergetic measurements after diamide and DTT addition to cells: After measurement of the baseline oxygen consumption (panel C) and extracellular acidification (panel D) rates (OCR and ECAR, respectively), diamide was injected to 0 (closed squares), 0.1 (triangles; dashed line), or 0.5 (circles; dotted line) mM concentrations. At the indicated time, DTT was injected to a final concentration of 1 mM, and OCR and ECAR measurements were recorded. N = 5 per group. (E) Cell viability in diamide and DTT-treated cells. Cells were treated with diamide in the absence or presence of 1 mM DTT for 16 h. Cell viability was then measured by MTT assay. N = 4 per group; *p < 0.05 vs. cells treated with diamide alone.