Pink1

The methods used for protein extraction from in vitro and in vivo samples and for western blot analysis have been described previously (Yu et al., 2017 (link)). The antibodies used include BNP (Abcam, ab236101, 1:500), MHC (Abcam, ab180779, 1:1000), Vinculin (Abcam, ab129002, 1:500), p62 (Cell Signaling Technology, #5114, 1:1000), LC3 (Abcam, ab48394, 1:500), β-actin-HRP (Kang Cheng, KC-5A08, 1:5000), VDAC1 (Abcam, ab154856, 1:1000), Ubiquitin (PTM BIO, PTM-1107, 1:2000), PINK1 (Abcam, ab23707, 1:500), Parkin (Abcam, ab77924, 1:500), FUNDC1 (Abcam, ab224722, 1:500), PHB2 (Santa Cruz, sc133094, 1:1000), BNIP3L (Cell Signaling Technology, #12396, 1:1000), p-Parkin (Ser65) (Affinity Biotech, AF3500, 1:1000).

Proteins from hippocampal tissues or cell lysates (20–40 μg of total protein) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane, and blocked with 5% BSA/Tris-buffered saline with Tween-20 (TBST) for 1 h at room temperature. Then, the membranes were incubated overnight at 4°C with primary antibodies at the following dilutions: NLRP3 (1:1000; abcam), caspase-1 (1:200; Santa cruz), ASC (1:1000; abcam), Bax (1:1000; CST), Bcl-2 (1:2000; CST), caspase-3 (1:1000; CST), Parkin (1:1000; CST), PINK1 (1:1000; abcam), LC3 (1:1000; CST), ATG5 (1:1000; CST), ATG7 (1:1000; CST), P62 (1:1000; CST), Beclin-1 (1:1000; CST), TOM20 (1:500; Beyotime), and GAPDH (1:500; Beyotime), followed by incubation with appropriate secondary antibodies after thoroughly washing three times with TBST. The bands were visualized using the chemiluminescence (ECL) detection system (Thermo Fisher Scientific) and quantified by Image J gel analysis software. Expression levels were normalized against GAPDH.

Melatonin, TBHP and type II collagenases were acquired from Sigma‐Aldrich (St Louis, MO). The primary antibodies of Collagen II, Aggrecan, Sox‐9, MMP‐13, ADAMTS‐5, Bax, Bcl‐2, Cytochrome C, P62, PINK1, Parkin, GAPDH and SOD2 were obtained from Abcam (Cambridge, MA, USA). The LC‐3, Beclin‐1 and Cleaved‐caspase 3 antibodies were acquired from CST (Beverly, MA). The FITC‐labeled and horseradish peroxidase‐labeled secondary antibodies were ordered from Bioworld (MN, USA). The 4', 6‐diamidino‐2‐phenylindole (DAPI) was acquired from Beyotime (Shanghai, China). The cell culture reagents were acquired from Gibco (Grand Island, NY).

MTT was purchased from Sigma‐Aldrich (USA). Chloroquine diphosphate, E‐64, PKI‐402 and rapamycin were purchased from MedChemExpress (USA). The primary antibodies against LC3B, SQSTM1/p62, TFEB, PINK1, parkin, Fis1, Drp1, Mfn1 and Mfn2 were purchased from Abcam (USA), and LAMP1, beta‐actin, alpha‐tubulin, cathepsin B, cathepsin D and cytochrome c were purchased from Proteintech (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.

A radio-immunoprecipitation assay lysis buffer, phenylmethanesulfonyl fluoride, and phosphatase inhibitor cocktail (Beyotime) were utilized to isolate proteins from cells and exosomes. The protein concentrations were evaluated by the BCA Protein Assay Kit (Beyotime) and adjusted. Proteins were separated and transferred onto polyvinylidene difluoride membranes (Millipore). Five percent non-fat powdered milk (Beyotime) was then used to block membranes for 1 h. After that, the membranes were incubated with different primary antibodies against LC3 (1:1000, Abmart), beclin1 (1:2000; Proteintech), Parkin (1:1000; ABclonal), PINK1 (1:1000; Abcam), CD206 (1:1000; Boster), Arginase-1 (Arg-1, 1:1000; Proteintech), CD63 (1:1000; UMIBIO), TSG101 (1:1000; UMIBIO), Aβ oligomer (1:1000, Abcam),Aβ1-42(1:1000,CST) separately overnight at 4°C. Afterward, members were incubated with corresponding secondary antibodies (Beyotime) for 1 hour at room temperature. The enhanced chemiluminescence (Tanon) was used to observe the blots. ImageJ software was used to quantify each band density.