L lysine

C2C12 cells were cultured on glass coverslips coated with L-Lysine (Nacalai Tesque). On day four of differentiation, myotubes after each treatment were fixed in 4% paraformaldehyde/phosphate buffer solution (PBS). C2C12 myotubes were permeabilized in 0.25% Triton X-100/PBS and blocked in 1% bovine serum albumin/PBS (blocking solution) for 1 h at room temperature. Myotubes were washed with PBS and incubated for 1 h in anti-muscle MHC antibody at a dilution of 1:250, washed with PBS, and incubated in Alexa Flour 488 conjugated goat anti-mouse antibody (Cell Signaling Technology, #4408) for 1 h, and washed with PBS. Antibodies were diluted in blocking solution. The samples were mounted in a 4’, 6-diamidino-2-phenylindole (DAPI)-containing mounting medium. Images were captured with a BZ-9000 fluorescent microscope (Keyence, Osaka, Japan). Representative images for each treatment were selected. The myotube width was measured by BZ II Analyzer software (Keyence). One hundred myotubes per treatment were measured from ten non-overlapping fields-of-view, and were used to evaluate myotube atrophy.