Nitroblue tetrazoliumand

To assess skeletal muscle myofiber metabolic phenotype succinate dehydrogenase (SDH) activity was measured. SDH is an enzyme bound to the inner mitochondrial membrane and SDH activity is higher in type 1 fibers [37 (link)]. TA tissue cryosections were taken out of the −80 °C and air dried on the laboratory bench and then incubated in an SDH staining solution containing 0.5 mg/mL nitroblue tetrazoliumand (Sigma, Cat#: N5514) and 50 mM sodium succinate (Sigma-Aldrich, St. Louis, MO, USA Cat#: S2378) in PBS for 30 min at 37 °C [38 (link)]. Slides were then washed in distilled water 3 × 1 min before mounting with warmed glycerol mounting media and cover slipped. Images of SDH stains were taken at 4×, 20×, and 40× using a Keyence BZX800 microscope. SDH activity was analyzed by measuring the circumference and mean grey value of ~300 fibers from throughout the entire muscle. Images were inverted thus higher values corresponded with higher SDH activity.

Total proteins were harvested from leaf samples as described previously by Sun et al. (2018) using 2× SDS loading buffer (100 mm Tris (pH 6.8), 4% (w/v) SDS, 20% (v/v) glycerol and 0.2% (w/v) bromophenol blue) containing 10% β-mercaptoethanol. Proteins were isolated on 12.5% SDS polyacrylamide gels and then transferred onto polyvinylidene fluoride membranes (Ge Healthcare, Buckinghamshire, UK). The membranes were blotted with a rabbit anti-flag antibody (1:1000; Sigma–Aldrich, St. Louis, MO, USA), mouse anti-flag antibody (1:5000; Sigma–Aldrich), rabbit anti-GFP antibody (1:3000; Genscript, Nanjing, China) or rabbit anti-PLRV MP antibody (1:5000) [53 (link)]. Immunoreactive proteins were successively visualized by blotting with goat anti-rabbit AP antibody (1:10,000; Sigma–Aldrich), goat anti-mouse AP antibody (1:10,000; Sigma–Aldrich), goat anti-rabbit HRP antibody (1:3000; Sigma–Aldrich) or goat anti-mouse HRP antibody (1:3000; Bio-Rad, Hercules, CA, USA) followed by visualization using nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolyphosphate (Sigma–Aldrich) or a chemiluminescence detection kit (GE Healthcare).

To identify which muscle types comprise the quadriceps in WT and TG mice, ATPase analysis was conducted using a ATPase Stain kit (#30-30125LY, Bio-Optica, Milano, Italy) following the manufacturer’s instructions. Briefly, cryostatic muscle sections (7 µm) of mice quadriceps were warmed up at room temperature for 10 min. The sections were incubated in each of the acetate buffers (pH of 4.3 and 10.4) for 10 min and Adenosine 5’-triphosphate solution for 30 min at 37 °C. After this, they were incubated in a cobalt chloride solution and an ammonium sulfide solution before being dehydrated and cleaned. Furthermore, in order to distinguish which muscles of WT or TG had oxidized to a greater degree, the quadriceps were stained by succinate dehydrogenase (SDH). Cryostatic muscle sections (7 µm) of mice quadriceps were warmed up and incubated in an SDH staining solution that consisted of 50 mM sodium succinate (#S2378, Sigma-Aldrich, St. Louis, MO, USA), 50 mM phosphate buffer (Welgene, Daegu, Korea) and 0.5 mg/mL nitroblue tetrazoliumand (#N5514, Sigma) for 40 min at 37 °C. All slides were visualized using an Olympus DP73 microscope (Olympus, Center Valley, PA, USA).