Coenzyme q2

Coenzyme Q2 and Q6 were purchased from Sigma Aldrich (#C8081, #C9504). Coenzyme Q9 was purchased from Cayman Chemical (#16866). Coenzyme Q10 was generously provided by Kaneka Corporation. Human Aβ_25–35 (#AS-24227) and Human Aβ_25–35 HiLyte™ Fluor 488-labeled (#AS-63308) were obtained from Anaspec. The fluorescent probes, Fluo-4 AM (#F23917), H₂DCF-DA (#C6827), MitoSOX (#M36008), MitoTracker Red CMXRos (#M7512), Calcein-AM (#C34852), and Hoescht 33258 (#861405), were obtained from Thermo Fisher. Ethidium bromide (EtBr, #46067) was acquired from Sigma–Aldrich.

NADH dehydrogenase activity was measured in 1 mL NDH buffer (50 mm potassium phosphate buffer, pH 7.5, 1 mm EDTA, 0.2 mm KCN), containing 20–30 μg proteins from the mitochondrial lysates and 5 μL of 20 mm NADH (AppliChem). After the addition of 2 μL 10 mm coenzyme Q₂ (Sigma-Aldrich), the change in absorbance at 340 nm was followed for 3 min (Čermáková et al., 2019 (link)). A unit of activity was defined as the amount of enzyme that catalyses the oxidation of 1 nmol NADH per min, assuming an extinction coefficient of 6.2 L mmol⁻¹ cm⁻¹ (Gonzalez-Halphen and Maslov, 2004 (link)). Solutions of the inhibitors were freshly prepared. Capsaicin (Sigma-Aldrich) was dissolved in ethanol, rotenone (Serva, Heidelberg, Germany) and DPI (diphenyl iodonium, Sigma-Aldrich) – in dimethylsulphoxide and methanol, respectively. Rotenone and DPI were added to the assay mixture immediately before the start of the reaction, Capsaicin was pre-incubated for 3 min. Native electrophoresis and in-gel activity staining methods were adapted from Zerbetto et al. (1997 (link)) and Wittig et al. (2007 (link)) and performed as described previously (Verner et al., 2014 (link)).

Aspirin, LPS, NAC, NADPH, reduced glutathione (GSH), 1-chloro 2,4-dinitrobenzene (CDNB), cumene hydroperoxide, glutathione reductase, 5,5’-dithio bis-2-nitrobenzoic acid (DTNB), cytochrome c, coenzyme Q2, antimycin A, dodecyl maltoside, N-nitrosodimethylamine (NDMA), erythromycin and ATP bioluminescent somatic cell assay kits were purchased from Sigma (St Louis, MO, USA). 2,7-Dichlorofluorescein diacetate (DCFDA) was purchased from Molecular Probes, Inc. (Eugene, OR, USA). Kits for mitochondrial membrane potential assays were procured from R & D Systems, MN, USA. Apoptosis detection kits for flow cytometry and IL6 and TNF-α measurement kits were purchased from BD Pharmingen (BD Biosciences, San Jose, USA). HepG2 cells were purchased from American Type Culture Collection (Manassas, VA, USA). Polyclonal antibodies against beta-actin, HO-1, IκB-α, NF-κBp65, PARP, Nrf-2 and cytochrome c were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Reagents for electrophoresis and Western blot analyses were purchased from Bio-Rad Laboratories (Richmond, CA, USA).