Coenzyme q1

Rotenone, 2-thenoyltrifluoroacetone, antimycin A, myxothiazol, potassium cyanide, 3,4-dehydro-l-proline, carboxin, methyl-succinate, l-proline, coenzyme Q1, doxycycline, N-acetyl cysteine, and o-aminobenzaldehyde were purchased from Sigma Aldrich, St. Louis, MO. Atpenin A5 was purchased from Santa Cruz Biotechnology, Dallas, Texas.

We assessed the ability of purified detergent-solubilized DsbB to reduce ubiquinone-1 (UQ1; coenzyme Q1; Sigma) in the presence of DsbA using a Cary 50 UV-visible spectrophotometer (Varian) (14 (link), 66 (link)). Reduction of UQ1 is accompanied by a decrease in its absorbance at 275 nm. The assay was performed in a final volume of 100 μl at 30°C in a reaction mixture of 30 μM reduced BpsDsbA (BpsDsbA_reduced), 1 μM BpsDsbB, and 45 μM UQ1 in 25 mM MES, 150 mM NaCl, 0.3% (wt/vol) DM, pH 6.5. Control reactions omitted BpsDsbA, BpsDsbB, or UQ1. The reaction mixtures with E. coli proteins had 30 μM EcDsbA_reduced, 80 nM EcDsbB, and 45 μM UQ1. Three independent experiments were conducted, and representative data from one of these independent experiments are reported.

Aliquots of coenzyme Q₁ (CoQ₁), coenzyme Q₁₀ (CoQ₁₀), decylubiquinone (DQ, Sigma-Aldrich, Rehovot, Israel), and idebenone (Idb, Santhera Pharmaceuticals, Liestal, Switzerland) were kept frozen as 10 mM stock solutions in DMSO. MTG (Molecular Probes, Eugene, Oregon, USA), TMRE and DCF (Biotium, Harvard, CA, USA) were diluted and stored according to the manufacturer's instructions. Unless otherwise stated, reagents were obtained from Sigma-Aldrich (Rehovot, Israel).

In all, 10 μl of mitochondrial suspension was added to 290 μl XNA (5 mM KH₂PO₄, 5 mM MgCl₂.6 H₂0 and 5 mg albumin) and 25 μl XNB (5 mM KH₂PO₄, 5 mM MgCl₂.6 H₂0, 5 mg human serum albumin and 7.1 mg saponin). This was incubated on ice for 5 min. In total, 72 μl of this mixture was added to 75 μl of Reagent 7 (2.33 ml master mix, 100 μl coenzyme Q1 (6 mM; Sigma-Aldrich C7956), 20 μl of Antimycin A (0.6 mg/ml; Sigma-Aldrich A8674) and 50 μl of KCN (20 mM)) then a blank was measured for 2 min at 340 nm. In all, 3 μl NADH (7.5 mM) was added and the absorbance measured for 2 min at 340 nm. In total, 2 μl rotenone (10% Sigma-Aldrich R8875) was added to inhibit complex I (control) and absorbance was measured for a final 2 min at 340 nm. NQR activity was calculated by adjusting for inhibition of complex I by rotenone and normalised to total mitochondrial protein.

Complex I activity was determined from the oxidation rate of NADH (Fluka) per mg protein. Cell pellets were sonicated for 20 sec on ice in IME buffer (50 mM imidazole, 2 mM MgCl₂, 1 mM EDTA, Protease inhibitors) and 80 µg cell extract was added to reaction buffer [1 mM EDTA, 50 mM KCl, 1 mM KCN, 1.2 µM antimycin A, 10 mM Tris-HCl (pH 7.4)]. Just before measurement, 150 µM NADH and 100 µM coenzyme Q1 (Sigma), as an electron acceptor, were added. Absorbance at 340 nm was measured over 2 minutes using a spectrophotometer at 30°C. NADH oxidation not blocked by rotenone (a complex I inhibitor, 2.5 µM) was removed from the calculation to measure NADH oxidation occurring in complex I only.
To validate a role for complex I inhibition by phenformin, 0.5 mM methyl succinate (Sigma) was added to complete growth media with phenformin at the same time to observe if phenformin’s anti-cancer cell effects were reversed. methyl succinate serves as an alternate energy source that bypasses complex I in the electron transport chain. Cell death was measured 24 hours after treatment.

Sodium succinate, rotenone, cytochrome c, coenzyme Q₁, coenzyme Q₁₀, antimycin A, decylubiquinol, 2′,7′-dichlorofluorescin diacetate (DCFH-DA) and Rhodamine 123 were purchased from Sigma (St. Louis, MO, USA). Tris base and NADH were purchased from Amersco Inc. (Palm Harbor, FL, USA). 2,6-Dichlorophenolindophenol (DCPIP) was purchased from Merck & Co. Inc. (Kenilworth, NJ, USA). Bovine serum albumin (BSA) for protein quantification was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All the other reagents used were of reagent grade and prepared in double-distilled water.