Mttfa

Melatonin was obtained from Selleckchem (USA). D-Glucose was obtained from Sigma-Aldrich (St. Louis, MO, USA). NAC (N-acetyl-L-cysteine) was purchased from Beyotime Biotechnology (Shanghai, China). The primary antibodies against STAR, P450, AMPK, P-AMPK, COXIV, Cytc, NRF1, Beclin-1, ATG7, ATG12, ATG5 and LC3 were obtained from Cell Signaling Technology (Beverly, MA, USA); 3β-HSD, SIRT1, mtTFA, ATPB, PGC1-α and acetyl-Lysine were purchased from Abcam Biotechnology (Cambridge, MA, USA); BNIP3L, SOD2 and Tubulin 1-α were obtained from Beyotime Biotechnology (Shanghai, China); and LXR, GPX4, GPX5 and Actin were purchased from Proteintech Group (Wuhan, China). The goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from Zhongshan Gold Bridge Biotechnology Co. Ltd. (Beijing, China). Alexa Fluor 488 donkey anti-mouse Immunoglobulin G (IgG) (H + L), MitoTracker™ Green FM and MitoSOX™ Red Mitochondrial Superoxide Indicator were obtained from Invitrogen (Carlsbad, CA, USA). The goat anti-mouse IgG/Alexa Fluor/594 were purchased from Zhongshan Gold Bridge Biotechnology Co. Ltd. (Beijing, China). Membrane and Cytosol Protein Extraction Kit, Cell Mitochondria Isolation Kit, Reactive Oxygen Species Assay Kit and Mitochondrial membrane potential assay kit with JC-1 were obtained from Beyotime Biotechnology (Shanghai, China).

For extraction of mitochondrial and cytosolic proteins, a commercially available kit from Sigma-Aldrich (#MITOISO2, Saint Louis, MO, USA) was used. For staining of mitochondria, MitoProbe™ JC-1 (5,5ʹ,6,6ʹ-tetrachloro-1,1ʹ,3,3ʹ-tetraethyl-imidacarbocyanine iodide) (#M34152, Molecular Probes, Invitrogen, CA) was used. A luciferase-based assay kit was purchased from Promega Corp. (#G7570, Madison, WI). Parkin (#ab77924), TOM20 (#ab186735), TIM23 (#ab230253), PGC-1α (#ab191838), mt-TFA (#ab131607), LC3 (#ab51520), P62 (#ab56416), P62 phospho S349 (#ab211324), COX IV (#ab202554), NLRP3 (#ab214185), ASC (#ab175449), caspase-1 (#ab179515), cleaved caspase-3 (#ab49822), IL-1β (#ab9722), KIM-1 (#ab47635), Bax (#ab32503), Bcl-2 (#ab182858) and GAPDH (#ab181602) antibodies were purchased from Abcam (Cambridge, UK). The CellTiter-Glo assay and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining kit were obtained from Keygen Biotech (#KGA7037, Nanjing, China). ELISA kits to assay inflammatory cytokines (tumour necrosis factor-α [TNF-α, #1217202], interleukin-1β [IL-1β, #1210122], and interleukin-6 [IL-6, #1210602]) were obtained from Dakewe Biotech (Shenzhen, Guangdong, China). The immunohistochemical kits were obtained from Beyotime (#P0203, Shanghai, China), and all the other chemicals were purchased from Sigma-Aldrich (Saint Louis, MO, USA).

Western blotting was performed using a standard protocol. We used the following primary and secondary antibodies: PCNA (1:1000; ab92552, Abcam, Cambridge, MA, USA), CYCLIN D1 (1:1000; #55506, Cell Signaling Technology, Danvers, MA, USA), phospho-CDK2 (p-CDK2; 1:1000; #2561, Cell Signaling Technology, Danvers, MA, USA), CDK2 (1:1000; #18048, Cell Signaling Technology, Danvers, MA, USA), SIRT1 (1:1000; #9475, Cell Signaling Technology, Danvers, MA, USA), PGC1α (1:1000; NAP1-04676, Novus Biologicals, Littleton, CO, USA), NRF1 (1:1000; #46743, Cell Signaling Technology), mtTFA (1:1000; ab131607, Abcam, Cambridge, MA, USA), ACTIN (1:2000; sc-8432, Santa Cruz Biotechnology, Dallas, TX, USA), ACTB (1:2000; A5316), HRP-conjugated anti-rabbit IgG (1:5000; #65-6120, Invitrogen, Carlsbad, CA, USA) and anti-IgG (1:5000; #62-6520, Invitrogen, Carlsbad, CA, USA). Bands were detected using a ChemiDoc XRD+ system (Bio-Rad, Hercules, CA, USA), and relative intensity was assessed using Image Lab software (Bio-Rad, Hercules, CA, USA).