Parkin

Vasicinone (purity > 98%) was purchased from Cayman Chemical (CAS-486-64-6, Ann Arbor, MI, USA). Paraquat, a Mitochondria Staining Kit (JC-1 stain) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The MitoSOXTM Red kit (M36008) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Dulbecco’s modified Eagle medium (DMEM:F12) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Primary antibodies against SOD-1, SOD-2, GST, GPx, TOM-20, VDAC-1, Parkin, PINK-1 and GAPDH were purchased from Santa Cruz Technology (Dallas, TX, USA), antibodies against DJ-1, α-synuclein, p-ULK, ATG7, ATG12 and LC3B were purchased from Cell Signaling Technology (Danvers, MA, USA) and an antibody against Nrf-2 was purchased from Abcam (Cambridge, MA, USA). All fluorescent secondary antibodies were purchased from Thermo Fisher Scientific in the USA.

Antibodies against PGC1α (Ab3243; Millipore, Billerica, MA), transcription factor A, mitochondrial (TFAM; Abcam Ab119684), NEF2L2 (Abcam ab31163), ClpP (Sigma HPA010649), Htra2 (AF1458; R&D Systems, Minneapolis, MN), mtHsp70 (Thermo Fisher Scientific), Hsp60 (611562; BD Biosciences, Franklin Lakes, NJ), GPS2,21 p62 (Progen GP62‐C), Beclin1 (Millipore Ab15417), Pink1 (Abcam Ab23707), LC3‐II (4108; Cell Signaling Technology, Danvers, MA), and parkin (sc‐32282; Santa Cruz Biotechnology, Santa Cruz, CA) to assess retrograte signaling factors and mitophagy markers were used in combination with MTCOI, SDHA, and DAPI to assess changes in relation with COX deficiency in single muscle sections. Antibodies that did not produce a sufficient signal‐to‐noise ratio, or were highly correlated with mitochondrial mass, were removed from further analysis. TFAM, Hsp60, GPS2, and Beclin1 were selected for immunofluorescence on serial sections (n = 4) of patients with multiple mtDNA deletions (n = 3, Patients 4, 5, and 7; see Supplementary Table). IMARIS v.7.7.2 (Bitplane) was used to assess average fluorescent intensity of signaling markers relative to MTCOI/SDHA ratio in whole COX‐positive and COX‐deficient fibers. Average intensity of the foci was compared to a COX‐positive region of the fiber.