Fig 2: Intermolecular FRET biosensors of mitochondrial respiratory chain complex detect reduced supercomplex formation by suppressing Cox7rp expression.a Knockdown of Cox7rp expression with siCox7rp in C2C12 myoblastic cells was performed by reverse transfection method. Two days after transfection, total RNA was extracted and knockdown efficiency was evaluated using qRT-PCR. Two different siRNAs (10 nM) targeting Cox7rp (siCox7rp #1 and #2) and two different siRNAs (10 nM) not targeting human transcripts (siControl #1 and #2) were used. Data are presented as means ± SE (n = 3 biologically independent samples). ***P < 0.001; two-way ANOVA. b Knockdown efficiency of COX7RP in C2C12 myoblastic cells evaluated by western blot analysis. Knockdown of Cox7rp expression with siCox7rp in C2C12 myoblastic cells was performed by reverse transfection method. Two days after transfection, the cells were lysed and subjected to western blot analysis with the COX7RP antibody. FP70 protein was blotted as an internal control. This experiment was repeated twice and the results of one experiment are shown. IB, immunoblot. c Mitochondrial proteins of C2C12 myoblastic cells treated with siCox7rp #1 or #2, or siControl #1 or #2 were solubilized and subjected to blue native polyacrylamide gel electrophoresis (BN-PAGE). Positions corresponding to mitochondrial supercomplex I/III2/IVn, I/III2/IV, I/III2, III2/IV, complex I, and dimerized complex III (III2) are indicated. BN-PAGE was performed with antibodies for NDUFB8 of complex I and UQCRC2 of complex III. FP70 protein was blotted as an internal control. This experiment was repeated twice and the results of one experiment are shown. d Fluorescence microscopy images of C2C12 myoblastic cells stably co-expressing NDUFB8-AcGFP and COX8A-DsRed-Monomer, treated with siCox7rp or siControl in live cells. FRET efficiency is shown by pseudo-color images. Indicated area (white square) are shown in the enlarged images. Scale bars, 10 µm. e–g Quantification of fluorescence intensities of C2C12 myoblastic cells stably co-expressing NDUFB8-AcGFP and COX8A-DsRed-Monomer, treated with siCox7rp or siControl by imaging cytometer. Donor intensities (e), acceptor intensities (f), and corrected FRET (cFRET)/donor ratios (g) are shown. Data from 12 wells are presented as means ± SE. n.s. not significant. ***P < 0.001, two-way ANOVA. Source data are provided as a Source Data file.

Fig 3: SYK inhibitors promote mitochondrial respiratory chain supercomplex assembly and stimulates oxygen consumption in C2C12 myoblastic cells.a Chemical structures of BAY61-3606 and GSK143. b, c cFRET/donor ratio of C2C12 cells stably co-expressing NDUFB8-AcGFP and COX8A-DsRed-Monomer treated by different concentrations of BAY61-3606 (b) and GSK143 (c). Data are presented as means ± SE of three wells for each treatment. d BN-PAGE of mitochondrial proteins from C2C12 cells treated with indicated SYK inhibitors (1 µM each) or DMSO for 24 h. Positions corresponding to indicated mitochondrial supercomplexes and dimerized complex III (III2) are indicated. Immunoblot (IB) was probed with anti-UQCRC2. FP70 was analyzed as an internal control. e IB for a mitochondrial fraction of C2C12 cells treated with indicated reagents as panel d, probed with antibodies against distinct respiratory complexes. f SDS-PAGE of whole cell lysates from C2C12 cells with treatment as panel d. IB was probed with indicated antibodies. ß-Actin was analyzed as an internal control. g, h Oxygen consumption rate (OCR) measurement of C2C12 cells treated with BAY61-3606 (g) or GSK143 (h) (1 µM each) and DMSO for 24 h. Data are presented as means ± SE from three biologically independent experiments. Basal respiration, ATP synthesis component of respiration, and maximal respiration were calculated as described in “Methods”. FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone. *P < 0.05; **P < 0.01; unpaired two-sided Student’s t test. Source data are provided as a Source Data file.

Fig 4: Intermolecular FRET biosensors of mitochondrial respiratory chain complex detect supercomplex formation in fixed and live cells.a Schema of five molecular complexes involved in oxidative phosphorylation; complex I–IV and ATP synthase. A subset of mitochondrial respiratory chain complexes forms a higher-order structure called supercomplex. The most abundant supercomplex consisting of complex I (CI) monomer, complex III (CIII) dimer, and complex IV (CIV) monomer (I/III2/IV) is shown. Förster resonance energy transfer (FRET) biosensors of AcGFP tethered to NDUFB8 (a subunit of CI) and DsRed-Monomer tethered to COX8A (a subunit of CIV) are shown. b, c Fluorescence microscopy images of C2C12 myoblastic cells stably co-expressing NDUFB8-AcGFP and COX8A-DsRed-Monomer (upper panels), and UQCR11-AcGFP and ATP5F1c-DsRed-Monomer (lower panels), in fixed (b) and live (c) cells. FRET efficiency is shown in pseudo-color image. These experiments were repeated twice and the results of one experiment are shown. Scale bars; 10 µm. d Fluorescence images of acceptor-photobleaching. In C2C12 myoblastic cells stably expressing NDUFB8-AcGFP and COX8A-DsRed-Monomer, DsRed-Monomer was partially photobleached by illumination at 558 nm for 3 min. The photobleached area (purple square) is indicated. Scale bars; 10 µm. e Fluorescence images of DsRed-Monomer and AcGFP channels before and after photobleaching. Fluorescence intensities were measured in selected regions (n = 9) and compared before and after bleaching. Data are presented as means ± SE. ***P < 0.001; paired two-sided Student’s t test. Source data are provided as a Source Data file.

Fig 5: Inhibition of Syk expression increases mitochondrial respiratory chain supercomplex assembly in C2C12 myoblastic cells.a Knockdown of Syk expression in C2C12 cells transfected with its specific siRNAs (siSyk #1 and #2) evaluated by qRT-PCR. Indicated siRNAs (100 pM each) were used. Data are presented as means ± SE of three biologically independent samples. ***P < 0.001; two-way ANOVA. b Immunoblotting (IB) for mitochondrial protein expression in C2C12 cells transfected with indicated siRNAs (100 pM each), probed with antibodies against distinct respiratory complexes. ß-Actin was analyzed as an internal control. c Imaging cytometer-based quantification of cFRET/donor ratios for C2C12 cells stably co-expressing NDUFB8-AcGFP and COX8A-DsRed-Monomer transfected with indicated siRNAs. Data are presented as means ± SE from 12 wells. ***P < 0.001; two-way ANOVA. d BN-PAGE of mitochondrial proteins from C2C12 cells treated with indicated siRNAs (100 pM each). Positions corresponding to indicated mitochondrial supercomplex and dimerized complex III (III2) are indicated. Immunoblot (IB) was probed with anti-UQCRC2. FP70 was analyzed as an internal control. e, f Oxygen consumption rate (OCR) measurement of C2C12 cells transfected with indicated siRNAs for 48 h. Data are presented as means ± SE from three biologically independent experiments. Basal respiration, ATP synthesis component of respiration, and maximal respiration were calculated as described in “Methods”. FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone. *P < 0.05; **P < 0.01; unpaired two-sided Student’s t test. Source data are provided as a Source Data file.