*2.2. Animals and Sample Collection*

The design for the feeding experiment is shown in Table S1. Briefly, a total of 30 Duroc × Landrace × Large White, male, weaned piglets with similar weights were divided randomly into 2 groups from days 0 to 14: the control group was fed a diet containing 1.31% Lys (n = 12), and the Lys deficiency group was fed a diet containing 0.83% Lys (n = 18). On day 15, six piglets closest to the average weight of each group were selected to determine skeletal muscle growth. Then, the remaining piglets in the Lys deficiency group were divided randomly into two groups from days 15 to 28: the Lys deficiency group was fed a diet containing 0.83% Lys (n = 6), and the Lys rescue group was fed a diet containing 1.31% Lys (n = 6). In addition, the remaining piglets in the control group were fed a diet containing 1.31% Lys between days 15 and 28 (n = 6). On day 29, all piglets were slaughtered, and the weight of their skeletal muscle was measured. Longissimus dorsi muscle samples were collected from all of the piglets at days 15 and 29, flash-frozen with liquid nitrogen and stored at − 80 ◦C.

### *2.3. Amino Acid Detection*

To determine the content and concentration of amino acids in longissimus dorsi muscle, samples containing 20 mg protein were weighed and hydrolyzed with 6 mol/L hydrochloric acid (HCL) at 110 ◦C for 22 h. Then, the hydrolyzed liquid was transferred into a 50 mL volumetric flask with ultrapure water. Then, 1 mL of hydrolyzed liquid was dried by distillation and re-dissolved in 0.02 mol/L HCL. Finally, the amino acid composition was analyzed by an amino acid analyzer (Hitachi L-8900, Tokyo, Japan).

#### *2.4. Protein Extraction*

Tissue samples (n = 3) were excised and transferred into new tubes containing tissue lysis buffer (1% SDS, 8 mol/L urea) and 1 mmol/L phenylmethanesulfonyl fluoride (PMSF, Sigma-Aldrich, St. Louis, MO, USA). Then, the lysates were homogenized for 4 min using a TissueLyser (CK1000, Thmorgan, Beijing, China) and incubated on ice for 30 min. The lysates were centrifuged at 12,000× *g* and 4 ◦C for 15 min, and the supernatants were collected. The concentration of proteins was quantified using a micro-bicinchoninic acid assay (BCA) kit (Thermo-Fisher, Waltham, MA, USA) and separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels.

#### *2.5. iTRAQ Proteome Analysis*

Proteins were treated with tris-(2-carboxyethyl)-phosphine (TECP, Sigma-Aldrich, St. Louis, MO, USA) and iodoacetamide and digested with trypsin. Then, the peptide mixture was labeled using the 8-plex iTRAQ reagent according to the manufacturer's instructions (Applied Biosystems, Foster City, CA, USA). Because there were eight samples, the peptides were divided into two parts for subsequent detection. For the first peptide group, the control group samples were labeled 115/116, the Lys deficiency group samples were labeled 117, the Lys rescue group samples were labeled 118/119, and the mixture (total of nine samples) was labeled 121. For the second peptide group, the control group samples were labeled 115, the Lys deficiency group samples were labeled 116/118, the Lys rescue group samples were labeled 119, and the mixture (total of nine samples) was labeled 121. Then, equal amounts of peptides from each peptide group were mixed together and vacuum dried.

Then, the peptides were separated by ultra-performance liquid chromatography (UPLC) with a Nano Aquity UPLC system (Waters, Milford, MA, USA) and analyzed in combination with a quadrupole-orbitrap mass spectrometer (Q-Exactive, Thermo-Fisher, Waltham, MA, USA) and an Easy-nLC 1200 (Thermo-Fisher, Waltham, MA, USA) for Nano LC-MS/MS analysis. Finally, the MS/MS data were searched using Protein Discoverer Software 2.1 against the Sus scrofa musculus database (UniProt, https://www.UniProt.org). The false discovery rate (FDR) applied to the control peptide level was defined as lower than 1%. For quantitative analysis, the 0.66 < fold change < 1.5 and *p*-value < 0.05 were the threshold values used to identify the differentially expressed proteins.

All identified proteins were annotated and classified by Gene Ontology (GO, http://www. geneontology.org), and the differentially expressed proteins were then analyzed by GOATOOLS 0.6.5 (https://pypi.org/project/goatools/) for the GO enrichment analysis. Data are available via ProteomeXchange with identifier PXD016396.

#### *2.6. Immunohistochemical Analysis*

First, the muscle samples were dehydrated with a 20% sucrose solution for 24 h and embedded in Tissue Tek to prepare the cryosections (5 μm, with at least six sections collected from each sample). Then, the tissue slides were incubated with Pax7 (MAB1675, R&D, Minneapolis, MN, USA) and Ki67 (NB500-170, Novus, Miami, FL, USA) at 4 ◦C overnight. After the slides were washed three times with phosphate-buffered saline (PBS), they were incubated with Alexa Fluor® 488 AffiniPure goat anti-mouse IgG (115-545-003, Jackson, West Grove, PA, USA) and Cy3-AffiniPure Goat anti-rabbit IgG (111-165-045, Jackson, West Grove, PA, USA) at room temperature for 90 min. Next, the slides were washed 3 times

with PBS and incubated with 4 ,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 5 min. Images were obtained using an immunofluorescence microscope (Ti2-U, Nikon, Tokyo, Japan TYPE, COMPANY, CITY, COUNTRY).

#### *2.7. Western Blotting*

Protein was extracted from the longissimus dorsi muscle or SCs with lysis buffer (RIPA, BioTeke, Beijing, China) and PMSF (Sigma-Aldrich, St. Louis, MO, USA). Next, the samples were centrifuged at 12,000× *g* and 4 ◦C for 15 min, and the protein concentration was determined using a micro BCA protein assay kit (Thermo-Fisher, Waltham, MA, USA). A total of 10 μg of protein was separated on 8–10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred onto polyvinylidene fluoride membranes (PVDF, Millipore, Darmstadt, Germany). After blocking, the membranes were incubated with specific primary and second antibodies (Table S2). Immunoreactivity was detected using an electrochemiluminescence (ECL) Plus chemiluminescence detection kit (Millipore, Darmstadt, Germany) and a Fluor Chem M system (Protein Simple, Santa Clara, CA, USA). The band density was analyzed using ImageJ Analysis Software (https://imagej.nih.gov) after excluding the background density (n = 3). The results were confirmed by three independent experiments with three samples per treatment.

#### *2.8. Isolation and Culture of SCs*

The method used to isolate, purify and identify the SCs was performed as described previously with modification [23]. In this study, SCs were isolated from the longissimus dorsi muscle of 5-day-old Landrace piglets and cultured in Dulbecco's modified Eagle's Medium/Nutrient Mixture F-12 (DMEM/F-12, Thermo-fisher, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Thermo-fisher, Waltham, MA, USA) at 37 ◦C and 5% CO2. The medium was changed every 48 h.
