**2.2. Analysis of complex II isoform expression**

Analysis of complex II isoform expression was carried out as described previously [8]. Briefly, total RNA was isolated from cultured cells using TRIzol LS reagent (Invitrogen) according to the manufacturer's instructions. After removal of residual DNA by treating with DNAse, cDNA was synthesized from total RNA using ReverTraAce (Toyobo, Japan) and an oligo (dT) primer. DNA sequence of the Fp I subunit of the complex II was amplified using Taq DNA polymerase with the sense primer 5′tacggagcaggca 3′ and the antisense primer 5′aatggtggcgggac 3′ and that of the Fp II subunit was amplified with the sense primer 5′cggacagaggcg3′ and the antisense primer 5′aatggctggcgggat3′. The resulting PCR products were subjected to agarose gel electrophoresis, and the two isoforms were identified by the size of the PCR products in the ethidium bromidestained agarose gels scanned with the electronic transilluminator FAS III (Toyobo, Japan).

#### **2.3. Isolation of mitochondria**

Cells cultured in three 225 cm2 flasks were harvested by trypsinization. The cells were suspended in mitochondria preparation buffer (250 mM sucrose, 20 mM HEPES, 3 mM EDTA, and 1 mM sodium malonate), pH 7.5, and homogenized with a power-driven glass-teflon Potter-Elvehjem homogenizer (20 passes). The homogenate was centrifuged at 700 × g for 15 min to pellet the cell debris and nuclei. The resulting supernatant was centrifuged at 14,000 × g for 15 min to sediment the mitochondria. The pellet was washed thrice with the mitochondria isolation buffer without sodium malonate, resuspended in the same buffer, and stored on ice until analysis. Integrity of the mitochondria was analyzed by citrate synthase activity in the presence and absence of 0.05% sucrose monolaurate (SML) in the assay mixture.

#### **2.4. H2 O2 assay in isolated mitochondria**

Hydrogen peroxide-mediated oxidation of the fluorescent probe Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) to resorufin was used to determine the H<sup>2</sup> O2 generation rate of the isolated mitochondria as described previously [31, 32]. Since Amplex Red does not enter the mitochondria, polyethylene glycol-conjugated SOD (PEG-SOD), a membrane permeable form of SOD, was added to the reaction mixture to convert the O2 − produced in the mitochondrial matrix into H2 O2 , which is subsequently released to the cytosol. The assay was performed in a 96-well plate maintained at 25°C. The resorufin formation rate was measured in SpectraMax Plus spectroflourometer (Molecular Devices, Sunnyvale, CA) at an excitation wavelength of 540 nm and emission wavelength of 590 nm. The H<sup>2</sup> O2 generation rate was calculated using a standard curve (0–1000 nmol/ml). To eliminate the fluorescence increment due to nonspecific oxidation of Amplex Red, a parallel assay was performed in the presence of catalase, an enzyme that scavenges H2 O2 , and the result was obtained by subtracting the amount of H2 O2 produced in the presence of catalase from that in the absence of catalase.

H2 O2 generation assays were also performed in the presence of respiratory chain inhibitors-10mM nitropropionic acid (NPA), which blocks the dicarboxylate-binding site of complex II; 10 μM atpenin A5, which blocks the Q site of complex II; and 10 μM antimycin A, which blocks the Qi site of complex III in order to dissect the specific ROS-producing site of the respiratory chain.
