## 2 Materials and Methods
### Protein Extraction and Western Blot Analysis  
The SOL muscles were homogenized (1:10 _w/v_) in T-PER(r) Tissue Protein Extraction Reagent (#78510, ThermoFisher Scientific, Waltham, MA, USA) containing a Halt(tm) protease and phosphatase inhibitor cocktail (100\(\times\)) (#78440, ThermoFisher Scientific, Waltham, MA, USA) with a homogenizer stirrer (#HS-30E, DAIHAN Scientific, Seoul, Korea) using a tissue grinder (#SL_cw.011.102, SciLab(r) Korea, Seoul, Korea). Following the manual instructions, the homogenates were centrifuged at 10,000 \(\times\)\(g\) for 15 min at 4 \({}^{\circ}\)C in a Sorvall Legend Micro 17R Centrifuge (#75002444, ThermoFisher Scientific, Waltham, MA, USA) to obtain the upper cytosolic fraction. Protein concentration was assessed using the Pierce(tm) Coomassieie protein assay kit (#23200, ThermoFisher Scientific, Waltham, MA, USA). The supernatants were heated in a mixture of 4\(\times\) Bolt(tm) LDS sample buffer (#B0008, Invitrogen, Waltham, MA, USA) and 10\(\times\) Bolt(tm) sample reducing agent (#B0009, Invitrogen, Waltham, MA, USA) at 70 \({}^{\circ}\)C for 10 min and cooled on ice for 10 min. Equal amounts of 40 ug of the protein were loaded onto Bolt(tm) 4-12% Bis-Tris Plus Gels, 15-well (#NW04125 BOX, Invitrogen) for target proteins, except for LC3B, which was separated with Bolt(tm) 12% Bis-Tris Plus Gels, and onto 15-well (#NW00125 BOX, Invitrogen) for 1 h at room temperature (RT) in diluted 20\(\times\) Bolt(tm) MOPS Running Buffer (#B000102, Invitrogen, Waltham, MA, USA). After electrophoresis, the gels were transferred in diluted 20\(\times\) Bolt(tm) Transfer Buffer (#BT00061, Invitrogen, Waltham, MA, USA) to 0.45 um nitrocellulose membranes (#1620115, Bio-Rad) or 0.2 um PVDF membranes (#1620112, Bio-Rad, Hercules, CA, USA) and used to a hydrophobic and low molecular weight protein (LC3B) for 1 h at RT. The membranes were briefly stained with Ponceau S solution (#P7170, Sigma-Aldrich, St. Louis, MO, USA) to confirm the successful transfer and rinsed with pure water until the background is disappeared. Non-specific proteins were blocked for 60 min at RT in 5% non-fat milk or bovine serum albumin (BSA) with Tris-buffered saline containing 0.1% tween-20 (TBS-T). The membranes were then incubated with the designated primary antibodies overnight at 4 \({}^{\circ}\)C. The primary antibodies used were as follows: topoisomerase II-beta (TOP2B, #25330), citrate synthase (CS, #390693), paired box 7 (PAX7, #81975), myogenic differentiation 1 (MYOD1, #377460), myogenin (MYOG, #12732), muscle atrophy F-box (MAFbx/atropin-1, #166806), and muscle ring finger protein-1 (MuRF-1, #398608) from Santa Cruz Biotechnology; AKT (#9272), p-AKTThr308 (#13038), p-AKTSer473 (#4060), mammalian target of rapamycin (mTOR, #2983), p-mTORSer2448 (#5536), forehead box O3\(\alpha\) (FOXO3\(\alpha\), #12829), p-FOXO3\(\alpha\)Ser253 (#13129), Beclin-1 (BECN-1, #3495), ATG7 (#8558), and p62 (#23214) from Cell Signaling Technology; Cathepsin L (CTSL, #133641) from Abcam; LC3B (#L7543) from Sigma-Aldrich. The next day, the membranes were washed twice for 5 min in 1\(\times\) TBS-T and then incubated with the designated secondary antibodies conjugated with HRP for 1 h at RT. The secondary antibodies were goat anti-rabbit IgG (#G-21234) and goat anti-mouse IgG (#62-6520) from ThermoFisher Scientific. Next, the membranes were washed thrice for 5 min in 1\(\times\) TBS-T, and immunoreactive proteins were detected using Amersham ECL western blotting detection reagents (#RPN2209, Cytiva). The band images were acquired using a ChemiDoc(tm) XRS\({}^{+}\) Imaging System (#1708265, Bio-Rad), and the band intensities were quantified using the Image Lab(tm) software (version 6.0; #1709690, Bio-Rad).