Rotenone and antimycin
A (1 [micro]M each) were added to block complex I and III, respectively, in order to determine the OCR unrelated to mitochondrial oxygen consumption.
We demonstrated that exposure to 100 [micro] KI for 2 hours significantly increased mitochondrial superoxide production, enhanced by either 0.5 [micro]M Rotenone (an inhibitor of mitochondrial complex I) or 10 [micro]M Antimycin
A (an inhibitor of complex III) .
For determination of oxidative phosphorylation properties, the ATP synthase inhibitor oligomycin at 0.75 [micro]M, the uncoupler carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) at 1 [micro]M, and complex III inhibitor antimycin
A at 2 [micro]M were added to wells after 27, 63, and 108 minutes, respectively.
[beta]-Naphthoflavone (BNF), DMSO, catalase, ubiquinol, ADP, sodium succinate, NADH, cytochrome C, lauryl maltoside, oligomycin, 2,4-dinitrophenol (DNP), rotenone, antimycin
, CH223191, proadifen, and resveratrol were obtained from Sigma Chemical Co.
After 2 minutes, added respiratory substrates were as follows: pyruvate (5 mM)/malate (2.5 mM) for complex I, succinate (5 mM) for complex II + complex III, in the presence of inhibitor of complex I, and rotenone (200 nM) and inhibitor of complex III antimycin
A (13 nM).
Behling et al., "Antimycin
A and lipopolysaccharide cause the leakage of superoxide radicals from rat liver mitochondria," Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, vol.
Next, an electron transport chain (ETC) accelerator, p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (FCCP at 1.5 [micro]M), was added, and after 3 measurements were taken, mitochondrial inhibitors rotenone (1 [micro]M) and antimycin
(1 [micro]M) were added, and three final measurements were taken.
ROS production from mGPDH can reach the levels of ROS from complex III when inhibited with antimycin
A (the most potent source of ROS in mitochondria) .
Streptozotocin (STZ), reduced and oxidized glutathione (GSH/GSSG), 1-chloro 2,4-dinitrobenzene (CDNB), cumene hydroperoxide, glutathione reductase, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), NADH, NADPH, cytochrome c, coenzyme Q2, sodium succinate, antimycin
A, dodecyl maltoside, resorufin, 7-ethoxyresorufin, methoxyresorufin, Hoechst 33342, and ATP bioluminescent somatic cell assay kits were purchased from Sigma-Aldrich (St.
Then, sequential additions of oligomycin (750 nM), carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP; 1 [micro]M), and antimycin
A + rotenone (A/R; both 1 [micro]M) (all from Sigma-Aldrich) were added with measurements taken after each.
Superoxide radical production was generated after mitochondrial depolarization after induction by antimycin
A, thus blocking the electron transfer , while lipid peroxyl radicals were initiated by addition of Cumene hydroperoxide (CumOOH) .
The reaction chamber was filled with ~1 x [10.sup.6] indicated cells, and the oxygen consumption rate (OCR) of each respiratory complex was measured after addition of appropriate substrates, respiratory inhibitors (2 [micro]M rotenone, 2 [micro]M antimycin
A, 0.5 [micro]M oligomycin), and mitochondrial uncoupler FCCP (2 [micro]M).