*2.11. Flow Cytometry*

SCs were seeded at a density of 5 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/well in 6-well culture plates (Corning, Corning, NY, USA) to detect the cell cycle distribution. The cultivation process was carried out as described above and according to a method described previously [25]. After harvesting at 24, 48 and 72 h, the cells were fixed in 70% ice-cold ethanol at −20 ◦C for cell cycle analysis. Before the samples were analyzed by flow cytometry using a Becton Dickinson Fluorescence Activating Cell Sorter Aria (BD Biosciences, San Diego, CA, USA), the cells were centrifuged at 200× *g* and 4 ◦C for 5 min, re-suspended in 1 mL PBS, treated with 100 μL 200 mg/mL DNase-free RNase, incubated at 37 ◦C for 30 min, and treated with 100 μL 1 mg/mL propidium iodide (PI, Sigma-Aldrich, St. Louis, MO, USA) at room temperature (25 ◦C) for 10 min (n = 6).

#### *2.12. Protein Synthesis*

To measure protein synthesis, a nonradioactive technique called surface sensing of translation (SUnSET) was used [26,27]. In this study, 1 μg/mL puromycin (Millipore, Waltham, MA, USA) was added to all wells for an additional 30 min of culture, and puromycin was detected by western blotting with an anti-puromycin antibody (Millipore, Waltham, MA, USA, Table S2). The total protein concentration was determined by BCA (Thermo-fisher, Waltham, MA, USA).

#### *2.13. Immunofluorescence Staining*

SCs were cultured for 96 h for the proliferation rescue assay and differentiation rescue assay. First, SCs were fixed in 4% paraformaldehyde for 30 min and then permeabilized with 0.1% Triton-X-100 for 10 min. After blocking with 1% bull serum albumin (BSA) and 10% goat serum for 30 min, the SCs were stained with primary antibodies for 90 min and then probed with goat anti-rabbit IgG (Table S2). In addition, the nuclei were labeled with 4 ,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA) for 5 min at room temperature. Images were obtained using immunofluorescence microscopy.

#### *2.14. Rapamycin Inhibition*

After Lys deficiency for 48 h, Lys rescue medium was added alone or combined with 20 or 50 nmol/L (nM) rapamycin for another 48 h. After a total of 96 h, cell viability was measured by MTT assay, and cell proliferation was measured by cell count assay. In addition, cell samples were collected to detect protein synthesis and the mTORC1 pathway by western blotting.

#### *2.15. Statistical Analysis*

The data were analyzed using Statistical Analysis System software (SAS, Version 9.2; SAS Institute, Cary, NC, USA). For control group and Lys deficiency group comparisons, the results were analyzed by t-test. For control group, Lys deficiency group and Lys rescue group comparisons, the mean data were assessed for significance using Tukey's test. The data are expressed as the mean ± S.E.M. Differences between treatments were considered statistically significant when *p* < 0.05 and extremely significant when *p* < 0.01.

#### **3. Results**

#### *3.1. Skeletal Muscle Growth in Piglets Relies on Dietary Lys Supplementation*

To determine the effects of dietary Lys supplementation on the skeletal muscle growth of weaned piglets, we developed the experimental design shown in Supplemental Table 1. After the piglets (initial body weight: control = 8.42 ± 0.11 kg versus Lys deficiency = 8.42 ± 0.08 kg) were fed the Lys-restricted diet for 14 d, we found that the growth of the piglets (final body weight: control = 11.91 ± 0.18 kg versus Lys deficiency = 11.33 ± 0.18 kg) was significantly suppressed (Table S4). In detail, compared with those of the control group, the relative weights of the longissimus dorsi muscle, extensor carpi radialis muscle, semimembranosus muscle, total forequarters muscle and total hindquarters muscle were significantly decreased by dietary Lys deficiency for 14 d (Table S4).

After dietary Lys deficiency for 14 d, the piglets received a diet we supplemented to match the level in the control diet for another 14 d. Obviously, the final weight of the piglets in the Lys rescue group was significantly increased compared with that of the piglets in the Lys deficiency group (Table 1). Moreover, compared with those after Lys deficiency for 28 d, the relative weights of the longissimus

dorsi muscle, lateral head of triceps of brachii muscle, extensor carpi radialis muscle, biceps femoris muscle, semimembranosus muscle, semitendinosus muscle, cranial tibial muscle, soleus muscle, lateral head of gastrocnemius muscle and total hindquarters muscle were all increased in the Lys rescue group, which was subjected to dietary Lys deficiency for 14 d and re-supplemented for another 14 d (Table 1). Collectively, these findings suggest that skeletal muscle growth in piglets relies on dietary Lys supplementation.

**Table 1.** Effect of dietary Lys re-supplementation on the skeletal muscle growth of weaned piglets on 28 d (n = 5, %) 1.


<sup>1</sup> Values without the same small letters within the same line indicate a significant difference (*p* < 0.05).
