*2.3. siRNA Transfection*

H9c2 cells were seeded for 40–60% confluency at 24 h. On the day of the experiment, cells were transfected using Lipofectamine RNAiMAX (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and FlexiTube small interfering RNA (siRNA, Qiagen, Germantown, MD, USA) according to the manufacturer's recommendations. Briefly, H9c2 cells were seeded with Opti-MEM ™ Reduced Serum Medium, GlutaMAX ™ (Thermo Scientific, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5% fetal bovine serum and 1% antibiotic solution to reach a 40–60% confluency in 24 h. On the next day, Lipofectamine RNAiMAX (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and FlexiTube siRNA mixtures were added. The following siRNA sequences (sense strand) were used: negative control (NC): UUC UCC GAA CGU GUC ACG, and ANT1: GAC GCA AAG CUU UCU UCA ATT. All experiments were conducted 48 h post-transfection.

Cell viability was determined by the trypan blue exclusion method using the TC20 Automated Cell Counter (Bio-Rad, Hercules, CA, USA).

#### *2.4. Mitochondrial Oxygen Consumption Rate and ATP Production*

Oxygen consumption rate and ATP production in H9c2 cells were determined using the Seahorse XFe24 analyzer (Agilent, Santa Clara, CA, USA). An equal number of cells were seeded and transfected at 24 h. Mitochondrial oxygen consumption rate and ATP production were determined 48 h post-transfection following manufacturer's recommendations. Briefly, cell media was changed to Seahorse XF DMEM Medium, pH 7.4, and supplemented with (in mM): 10 glucose, 1 sodium pyruvate, and 2 L-glutamine. Mitochondrial functional parameters were determined after the addition of (in μM): 0.5 oligomycin, 4 carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and 0.5 rotenone/antimycin A. Data were extracted using the Seahorse XFe24 report generator and normalized to total protein.

#### *2.5. Isolation of Mitochondria*

H9c2 cells were trypsinized and pelleted at 200× *g* for 7 min. The pellet was resuspended in ice-cold sucrose bu ffer containing (in mM): 300 sucrose, 10 Tris-HCl, and 2 EGTA; pH 7.4. Cells were centrifuged at 2,500× *g* for 5 min at 4 ◦C, the pellet was resuspended in sucrose bu ffer and incubated on ice for 5 min. To disrupt the plasma membrane and expose mitochondria, cells were plunged using a 27G needle, until all cells were successfully lysed. The cell lysate was then centrifuged at 400× *g* for 5 min and the supernatant was collected. The mitochondria were concentrated by centrifugation at 10,000× *g* for 5 min and the final pellet was dissolved in sucrose bu ffer.

To isolate liver mitochondria, the liver tissue removed from WT and *SIRT3* KO mice was cut and homogenized using a Polytron homogenizer in 2 mL of ice-cold sucrose bu ffer containing: 300 mM sucrose, 20 mM Tris-HCl, and 2 mM EGTA. Homogenate was then centrifuged at 2,000× *g* for 3 min, to remove cell debris. The supernatant was then centrifuged at 10,000× *g* for 15 min to precipitate mitochondria. The final pellet was washed once with sucrose bu ffer by centrifugation at 10,000× *g* for 10 min. The mitochondria-enriched pellet was resuspended in 200 μL of sucrose bu ffer.

#### *2.6. Enzymatic Activity of ETC Complexes in Cultured Cells*

Enzymatic activity of ETC complexes was determined as previously described [15], with minor modifications and normalized to mg of mitochondrial protein. All assays were performed at the SpectraMax Microplate Reader (Molecular Devices, San Jose, CA, USA) at 37 ◦C.

#### *2.7. Total and Mitochondrial ROS Production in Cultured Cells*

Total ROS and mtROS production were measured with <sup>2</sup>,7-dichlorodihydrofluorescein diacetate (H2DCFDA) and MitoSOX Red, respectively [15]. Briefly, cells were incubated for 30 min with 10 μM H2DCFDA or 1 μM of MitoSOX and fluorescence intensity was monitored on the SpectraMax Microplate Reader (Molecular Devices, San Jose, CA, USA) at the excitation/emission of 599 nm/522 nm (for H2DCFDA) and 510 nm/580 nm (for MitoSOX).

#### *2.8. Mitochondrial Membrane Potential and Total ATP*

To measure ΔΨm in cultured cells, H9c2 cells were incubated with ΔΨm-sensitive dye JC-1 (5,5,6,6-tetraethyl-benzimidazolylcarbocyanine iodide; Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA). Briefly, cells were incubated for 30 min at 37 ◦C with JC-1 and fluorescence was measured using a SpectraMax Microplate Reader (Molecular Devices, San Jose, CA, USA). J-aggregates (red) and JC-1 dye monomers (green) were monitored at 530 and 590 nm emission (with excitation at 488 nm), respectively. Data are presented as the ratio red/green fluorescence.

ATP levels were measured using the ATP Bioluminescence Assay Kit CLS II (Roche, Indianapolis, IN, USA), according to the manufacturer's recommendations. Luminescence data were normalized to total protein levels.

#### *2.9. SDS-PAGE and Western Blotting*

To analyze protein levels, equal amounts of protein were resolved by SDS-PAGE and transferred onto Amersham Hybond ECL nitrocellulose membranes (GE Healthcare Bio-sciences, Pittsburgh, PA, USA). The membranes were immunoblotted with antibodies against ANT1 (Abcam #110322, Cambridge, MA, USA), or ATP5a (Abcam #14748, Cambridge, MA, USA) followed by incubation with IRDye® (LI-COR Biosciences, Lincoln, NE, USA) secondary antibodies. Bands were visualized using an ODYSSEY® CLx (LI-COR Biosciences, Lincoln, NE, USA) infrared scanner. The resulting images were analyzed with Image Studio Lite Software version 5.2.
