*2.15. TTR Overexpression Vector*

The region corresponding to the TTR gene open reading frame (ORF) was PCR amplified with TTR ORF primer (Forward: 5 -ATGGCTTCCCTTCGACTCTTCC-3 , Reverse: 5 -GATTCTGGGGGTTGCTGACGA-3 ) and ligated into the pcDNA 3.1/CT-GFP-TOPO vector (Invitrogen, Waltham, MA, USA). The ligated sequence was confirmed by sequencing analysis. The construct (2.5 μg) was transfected using 10 μL lipofectamine (1 mg/mL) and Opti MEM medium (Invitrogen) into C2C12 cells following the manufacturer's directions and positive cells were selected using G418 antibiotics (2 μg/mL, AppliChem GmbH, Darmstadt, Germany).

#### *2.16. Immunocytochemistry*

The cells were fixed with 4% formaldehyde (Sigma Aldrich) and permeabilized with 0.2% Triton X 100 (Sigma Aldrich). After blocking with 1% normal goat serum (SeraCare Life Sciences, Milford, MA, USA) for 30 min in a humid environment, cells were incubated with primary antibodies [TTR (1:50), MYOD (1:50), MYOG (1:50), MYL2 (1:50), D2 (1:50), RXRγ (1:50), TRα (1:50), or FNDC5 (1:50)] at 4 ◦C in a humid environment overnight. Secondary antibody (1: 100; Alexa Fluor 594 goat anti-rabbit or anti-mouse; Thermo Fisher Scientific) was applied for 1 h at room temperature. DAPI was used to stain the cells (Sigma-Aldrich) and imaged using a fluorescence microscope equipped with a digital camera (Nikon, Tokyo, Japan).

#### *2.17. Immunohistochemistry*

The sections of paraffin-embedded muscle tissue were deparaffinized and hydrated with xylene (Junsei, Tokyo, Japan) and ethanol (Merck), respectively, and endogenous peroxidase activity was quenched in 0.3% H2O2/methanol. The sections were then either stained with hematoxylin and eosin (Thermo Fisher Scientific) for morphological observation or blocked with 1% normal goat serum (SeraCare Life Sciences), incubated with primary antibodies [TTR (1:50), D2 (1:50), or FNDC5 (1:50)] overnight at 4 ◦C, and then incubated with horse radish peroxidase–conjugated secondary antibody (1:100). Positive signals were visualized by adding horse radish peroxidase-conjugated streptavidin (Vector, CA, USA). Nuclei of stained sections were stained with hematoxylin and then dehydrated, mounted, and observed by a light microscope (Leica, Wetzlar, Germany).
