Coxii

Specific proteins were detected using rabbit polyclonal antibodies against: MRPL44, MRPL37, MRPL23, MRPS12, MRPS16 (Proteintech, diluted 1:1000), MRPS34 (Sigma, diluted 1:1000), GAPDH, phosphorylated and non‐phosphorylated S6, phosphorylated and non‐phosphorylated Akt, phosphorylated and non‐phosphorylated 4EBP (Cell Signalling Technology, diluted 1:500) and mouse monoclonal antibodies against: phosphorylated and non‐phosphorylated AMPKα (Cell Signalling Technology, diluted 1:500), COXII (Abcam, diluted 1:1000), phosphorylated and non‐phosphorylated YAP, AFG3L2, LC3A/B, Glut2, Glut4, and Total OXPHOS Rodent WB Antibody Cocktail (Abcam, Diluted 1:1000) in Odyssey Blocking Buffer (Li‐Cor). IR Dye 800CW Goat Anti‐Rabbit IgG or IRDye 680LT Goat Anti‐Mouse IgG (Li‐Cor) secondary antibodies were used and the immunoblots were visualized using an Odyssey Infrared Imaging System (Li‐Cor).

were Twinkle (mouse-monoclonal), kind gift from Prof. Anu Suomalainen-Wartiovaara (see also7 (link)); TFAM (rabbit-polyclonal), kind gift from Prof. Rudolf Wiesner; ATAD3 rabbit-polyclonal11 (link); coxII, Cyclophilin D, POLRMT, PHB1, HSP60 and Mitofilin, from Abcam; polG, coxII, TOM20, VDAC1 and FACL4, from Santa Cruz; MRPL49 and MRPS22, from Proteintech Europe; Calnexin from Cell signaling and SSB from Sigma Aldrich, Atlas.

Skeletal muscle and heart tissues were resuspended in lysis buffer (1% Tx-100, 10 mM Tris-HCI pH 7.6, 150 mM NaCl, 1 mM EDTA) with protease inhibitor and phosphatases (Roche Diagnostic) and then were centrifuged at 11,000× g for 15 min (4 °C). Protein concentration was quantified with Micro BCA Kit (Thermo Fisher Scientific, Waltham, MA, USA). Samples were incubated for 5 min in TC 4X buffer (β-Mercaptoethanol) at 95 °C and 30 µg of protein extracts were separated by SDS-PAGE electrophoresis in 12.5% polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (GH Healthcare, Chicago, CA, USA). The following antibodies were used for inmunodetection: NDUFB11 (Abcam, 1:1000, Cambridge, UK), NDUFA9 (Abcam, 1:1000), NDUFV1 (Santa Cruz Biotechnology, 1:1000, Dallas, TX, USA), NDUFB8 (Abcam, 1:500), Core2 (Abcam, 1:2000) and COXII (Abcam, 1:1000). SDHA (Abcam, 1:10,000) was used as loading control. Secondary antibodies conjugated to horseradish peroxidase (Cell Signaling Technologies, Danvers, MA, USA) were used to detect the primary antibodies and the reactions were developed with ECL Prime Western Blotting Detection Reagent (GE Healthcare, Amersham, UK) in a ChemiDoc™ MP Imaging System (Bio-Rad, Hercules, CA, USA). The optical densities of the immunoreactive bands were measured using NIH ImageJ software v1.8 (Wayne Rasband, NIH, Bethesda, MD, USA).

Isogenic HCT116 WT and HCT116 Bax^−/− colon cancer cells were kindly provided by Dr. B Vogelstein^{54 (link),55 (link)} and cultured in McCoy's 5A medium supplemented with 10% FBS. The primary antibodies against cytochrome c (monoclonal antibody, mAb) were purchased from BD Biosciences (San Jose, CA, USA). COX II (Abcam, Cambridge, MA, USA), Hsp60 (EMD Millipore, Cambridge, MA, USA), and actin (mAb; BD Biosciences) were obtained from the indicated suppliers. Anti-Tfam, rabbit monoclonal anti-DNA POLG, and anti-Twinkle antibodies were purchased from Abcam. MitoProfile Total OXPHOS human WB Antibody cocktail (Abcam) was used as primary antibodies for western blot analysis.
Secondary antibodies and ECL reagents were acquired from GE Healthcare (Pittsburgh, PA, USA). MitoTracker Green, MitoTracker Orange, CM-H₂XRos, MitoSox, and DHR123 were purchased from Thermo Fisher Scientific (Carlsbad, CA, USA). The fluorogenic caspase-3 substrate DEVD-AFC was obtained from Enzo Life Sciences (Farmingdale, NY, USA). All other chemicals were purchased from Sigma Chemical Company (St. Louis, MO, USA) unless specified otherwise.