Bnip3

The protein was extracted using RIPA lysis buffer (Sangon Biotech) added with protease inhibitor. The bicinchoninic acid protein determination method was used to detect the protein concentration. Then, 25 μg protein sample was taken, treated with sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane in transfer buffer. The membrane was sealed with 5% skim milk for 1 h, and then incubated overnight at 4°C with the following primary antibodies: KDM3A (1:2000; Novus Biologicals), BNIP3 (1:1000; Abcam) and GAPDH (1:500; Abcam). Thereafter, the membrane was incubated with horseradish peroxidase‐labelled secondary antibody IgG (1:1000; Abcam) for 2 h. The protein blot visualization was achieved through an ECL Plus Western blot analysis system. GAPDH was set as the internal control.¹³

Briefly, the mice organs were collected and disrupted in lysis buffer containing protease inhibitors (Roche Diagnostics, Berlin, Germany). After centrifugation, the supernatant was collected, and equivalent amounts of protein were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The protein bands were visualized using DyLight 800/DyLight 680-conjugated secondary antibodies, and an infrared fluorescence image was obtained using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). Western blot analyses were performed by ImageJ with anti-Gapdh (KM9002, Sungene, Tianjin, China), anti-Lc3b (SAB4200361, Sigma, St Louis, MO, USA), anti-Sqstm1 (PM045, MBL International, Japan), and anti-Eva1a (NB110-74787, Novusbio, Littleton, CO, USA) antibodies. Antibodies against Ulk1, Akt, Mtor, Erk1/2, Lkb1, Ampk, Rps6kb1, and Eif4ebp1 and against phosphorylated Ulk1, Akt, Mtor, Erk1/2, Lkb1, Ampk, Rps6kb1, and Eif4ebp1 were purchased from Cell Signaling Technology (Boston, MA, USA). Antibodies against Drp1, Tomm20, Pink1, Parkin, Bnip3, Mitofusin2, and Pgc1 were purchased from Abcam (Cambridge, UK). DyLight 800/DyLight 680-conjugated secondary antibodies against mouse or rabbit IgG were purchased from Rockland Immunochemicals (Limerick, PA, USA).

Protein extractions from FFPE tissues were performed using a Qproteome FFPE tissue kit (Qiagen) according to the manufacturer's manual. Briefly, two or three sections from the same block were deparaffinized in xylene and rehydrated in a graded series of ethanol (100%, 96%, and 70%). The tissues were mixed with FFPE extraction buffer containing β-mercaptoethanol, incubated at 100°C for 20 min, at 80°C for 2 h with agitation at 750 rpm, and then centrifuged for 15 min at 14,000 ×g at 4°C. Extracted protein content in the supernatant was determined by Bradford assay (Bio-Rad). Equal amounts of protein from each sample extract were separated on SDS-PAGE gels and blotted onto nitrocellulose membranes (Bio-Rad). Western blotting was performed with primary antibodies against beclin-1. LC3A, LC3B, p62, BNIP3, and GAPDH (Abcam) and specific bands were detected using an ECL kit (GE Healthcare Life Sciences).

D-(+)-Trehalose dehydrate (purity >99 %), toluidine blue, tert-Butyl hydroperoxide solution (TBHP), bafilomycin A1 (Baf), chloroquine (CQ) and type II collagenases were purchased from Sigma-Aldrich (St Louis, MO, USA). The primary antibody against p62, Tom20, BNIP3, PGAM5, Drp1, SOD2, PARP, cytochrome C, caspase 9, 8-0HdG and β-actin were from Abcam (Cambridge, UK), LC-3 antibody was from Sigma-Aldrich (St Louis, MO, USA). GRP78 and Calnexin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Anti-CHOP, goat anti-rabbit, and anti-mouse IgG-HRP were from Bioworld (OH, USA) and antibodies against Atg3, Atg7, Atg12, Beclin1, p-AKT, AKT, p-mTOR, mTOR, p-p70S6K, p70S6K, p-AMPK, AMPK, p-ULK1, ULK1, ubquintin, caspase 12, Bcl-2, Bax and cleaved-caspase3 were from Cell Signaling (Danvers, MA, USA); Alexa Fluor488 labeled and Alexa Fluor594 labeled Goat Anti-Rabbit IgG (H+L) second antibody were from Jackson ImmunoResearch (West Grove, PA, USA). 4′,6-diamidino-2-phenylindole (DAPI) was from Beyotime (Shanghai, China).

C2C12 myotubes were lysed in RIPA buffer containing protease inhibitor and PMSF to extract the total protein. Equal quantities of proteins (20 μg) were separated by 10%–12% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% nonfat milk and incubated with primary antibodies targeting HIF-1α (1 : 1000; Abcam, Cambridge, UK), BNIP3 (1 : 1500; Abcam), atrogin-1 (1 : 1000; Abcam), LC3B (1 : 1000; ABclonal, Woburn, MA), beclin-1 (1 : 2000; ABclonal), myogenin (1 : 500; Millipore, Billerica, MA), parkin (1 : 1000; CST, Danvers, MA), p62 (1 : 500; CST), p-mTOR (1 : 1000; CST), mTOR (1 : 1000; CST), p-AMPKα (1 : 1000; CST), and AMPKα (1 : 1000; CST) overnight at 4°C. The membranes were incubated with goat anti-mouse or anti-rabbit secondary antibody for 1 hour at room temperature. Band intensity was determined using a chemiluminescent imaging system (Tanon, Shanghai, China). Tubulin was used as a control for protein level quantification.

Whole-cell lysates were collected using RIPA buffer III (Bio Basic Inc., Markham, Ontario, Canada) and subjected to 12% SDS-PAGE for separation. Next, the gel was transferred onto a PVDF membrane (Pall, Ann Arbor, MI, USA) and blocked with 5% skim milk in TBST (0.05% Tween 20 in TBS). Primary antibodies against LC3 (MBL International, Woburn, MA, USA), phosphor Tyr 705-STAT3 (Cell Signaling Technology, Beverly, MA, USA), total STAT3 (Cell Signaling Technology), BNIP3 (Abcam, Cambridge, MA, USA), caspase 3 (Imgenex, San Diego, CA, USA), β-actin (Sigma-Aldrich, St Louis, MO, USA) or MIF rabbit polyclonal antibody purified from recombinant human MIF (rMIF)-immunized rabbit sera were incubated with membrane at 4 °C overnight and washed with TBST. HRP-conjugated goat anti-rabbit or rabbit anti-mouse IgG secondary antibodies (1 : 10 000 dilution; Leadgene Biomedical) were incubated for another 1 h and detected using an Enhanced Chemiluminescence Western Blotting Kit (Advansta, Menlo Park, CA, USA). The results of the western blotting were quantified using the Image J software (National Institutes of Health, New York, NY, USA).