Blocking one

Cells and skeletal muscle tissues were fixed with 4% paraformaldehyde in PBS for 15 min at 4°C, prior to embedding in Frozen Section Compound (Leica Microsystems) for cryosections. Fixed samples were incubated with 0.1%Triton X-100 in PBS for 5 min, BlockingOne (Nakacai Tesque) for 30 min, and anti-GFP (Molecular Probes A6455; diluted 1:500), anti-PAX3 (DSHB AB528426; diluted 1:100), anti-PAX7 (DSHB AB528428; diluted 1:100), anti-myogenin (DAKO M3559; diluted 1:100), anti-MyoD (Abcam ab64159; diluted 1:500), anti-laminin (Enzo Life Sciences ALX-804-190; diluted 1:500), anti-dystrophin (Abcam ab15277; diluted 1:200), anti-MyHC (R&D MAB4470; MF20, diluted 1:200), anti-FSP1 (Abcam ab124805; diluted 1:200), anti-lamin A/C (Abcam ab8984; diluted 1:200), and anti-Ki67 (Abcam ab15580; diluted 1:500) antibodies in 5% BlockingOne overnight at 4°C. After three washes with 0.1% Tween 20 in PBS, cells were incubated with Alexa-conjugated anti-mouse immunoglobulin G1 (IgG1), mouse IgG2b, rabbit IgG, or rat IgG antibodies (Molecular Probes; diluted 1:500). Cells were washed and mounted in ProLong Diamond antifade reagent with DAPI (Molecular Probes). Images were collected and processed by BZ-X710 microscopy.

Myotubes were incubated with 1 μM puromycin (EMD Millipore, Temecula, CA, USA) for 30 min. Lycates were prepared with Pierce M-PER (Thermo Fisher Scientific, Waltham, MA, USA) and protein concentration was measured for each lysate using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Five micrograms of protein were separated in a 10% polyacylamide gel (TEFCO, Tokyo, Japan) under denaturing conditions until the dye-front was ∼1.5 cm from the bottom of the gel. Proteins were transferred to PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA), and then incubated in Blocking One (Nacalai) and incubated overnight with a monoclonal antibody to puromycin (1:5,000 dilution, clone 12D10; Millipore EMD) or GAPDH (1:10,000 dilution, ab181602; Abcam, London, England) diluted in Blocking One. Membranes were then incubated with secondary IgG mouse antibody (1:20,000 dilution, ab197767; Abcam) or IgG rabbit antibody (1:10,000 dilution, NA934; GE Healthcare, Chicago, IL, USA). The total lane density was analyzed using Fusion FX (Vilber Lourmat, Collégien, France). Protein expression was normalized to that for GAPDH (loading control).

After amino acid starvation and re-stimulation, cells were rinsed twice with ice-cold PBS and mounted on dishes with glass bottom (Greiner Bio-One, Stuttgat, Germany) using SmearGell (GenoStaff, Tokyo, Japan). The embedded samples were fixed with 4% paraformaldehyde/PBS for 15 min at room temperature. They were then rinsed twice with PBS, and permeabilized with 0.5% Triton X-100 in PBS for 6 min. After rinsing twice with PBS, the samples were blocked for 40 min with Blocking-One (Nacalai Tesque, Kyoto, Japan), and then incubated with primary antibodies in Blocking-One overnight at 4°C, rinsed twice with PBS, and incubated with Alexa Fluor 488 and 594-conjugated secondary antibodies (Abcam, Cambridge, UK) for 30 min at room temperature. The cells were washed with PBS and DAPI (Sigma-Aldrich) for nuclear staining, and then rinsed with 0.1% Triton X-100. Images were acquired on a spinning disk confocal super-resolution microscope (SpinSR10, Olympus, Tokyo, Japan) with a 100 X oil immersion objective lens (Olympus). To quantify co-localization, Pearson’s correlation coefficients were calculated from approximately 50–100 cells per sample using Cellsens imaging software (Olympus) with manually set thresholds. All the images were obtained and analyzed in a single setting. The primary antibodies used were mTOR (CST #2983; 1:400) and LAMP1 (Santa Cruz Biotechnology #SC-20011; 1:400).