The N-terminal 15 residues of P. yezoensis peptide PYP1 (1–15) (D-P-K-G-K-Q-Q- A-I-H-V-A-P-S-F; designated as PYP1-5) were synthesized by Peptron (Daejeon, Korea). PYP1-5 purification was performed using a Prominence High-Performance Liquid Chromatography apparatus (Shimadzu Corporation, Kyoto, Japan) with a Capcell Pak C18 column (column dimensions, 150×4.6 mm; particle size, 2.7 µm; Shiseido Co., Ltd., Tokyo, Japan) in 0.1% trifluoroacetic acid (TFA; v/v in water), and an elution gradient of 10–70% acetonitrile (0–20% acetonitrile for 2 min; 20–50% acetonitrile for 10 min; 50–80% acetonitrile for 2 min) in 0.1% TFA, a flow rate of 1.0 ml/min and UV detection at 220 nm; analysis was conducted with the Class-VP software package, version 6.14 (Shimadzu Corporation). The molecular mass of PYP1-5 was confirmed to be 1,622 Da (Fig. 1) using an HP 110 Series LC/MSD mass spectrometer [ionization mode, positive; nitrogen flow, 7 l/min; high vacuum, 1.3E-5 torr; neb press, 40 psi; quadrupole temperature, 100°C; flow rate, 0.4 ml/min (isocratic ANC:DW=8:2, 0.1% TFA); Peptron, Inc., Daejeon, Korea].

C2C12 mouse skeletal muscle cells (ATCC no. CRL-1772; American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at a temperature of 37°C in a humidified atmosphere of 5% CO₂. The cells were cultured to 70–80% confluence in 100 mm dishes, and the medium was replaced every 2 days.

C2C12 myoblasts were grown to 70–80% confluence in culture dishes at 37°C, trypsinized and seeded (4×10⁴ cells/well) into 6-well culture plates for experiments. Cells were grown to 70–80% confluence in DMEM supplemented with 10% FBS at 37°C for 24 h, at which time the medium was replaced with DMEM containing 2% FBS to induce myotube differentiation; the medium was replaced every 2 days. Cells were allowed to differentiate for 6 days, at which point 90% of the cells had fused into myotubes.

Following 6 days of differentiation, C2C12 myotubes were treated with 1, 10, 50 or 100 µM DEX at 37°C for 24 h for concentration screening. The C2C12 myotubes were subdivided into four groups: i) the control group, in which cells were incubated in serum-free medium (SFM; DMEM containing 100 U/ml penicillin and 100 mg/ml streptomycin); ii) the DEX group, in which cells were treated with 100 µM DEX; iii) the DEX+PYP1-5 group, in which cells were treated with 100 µM DEX and 500 ng/ml PYP1-5; and iv) the PYP1-5 group, in which cells were treated with 500 ng/ml PYP1-5. All groups were incubated at 37°C for 24 h, prior to harvesting cells for experiments.

Cell viability was measured using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation assay (Promega Corporation, Madison, WI, USA), which is based on the cleavage of 3-(4,5-dimethythiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS) into a formazan product that is soluble in tissue culture medium, according to the manufacturer's protocol. Cells (1.5×10⁴ cells/well) were seeded in 96-well plates in 100 µl DMEM supplemented with 10% FBS and were allowed to attach at 37°C for 24 h. Attached cells were maintained in SFM at 37°C for 4 h and were subsequently treated with or without PYP1-5 (125, 250, 500 and 1,000 ng/ml) for 24 h. MTS solution (10 µl) was added and the cells were incubated at 37°C for 30 min; the absorbance of each well was measured at 490 nm using a SpectraMax 340 PC Microplate Reader (Molecular Devices, LLC, Sunnyvale, CA, USA). Experiments were performed in triplicate.

Images of myotube cultures were captured with a phase contrast microscope set to ×100 magnification at 1, 2, 4, 5 and 6 days following induction of differentiation. Images of myotube cultures were also captured following treatment with 100 µM DEX and/or 500 ng/ml PYP1-5 for 24 h. A total of 50 myotube diameters from at least 10 random fields were measured using ImageJ software (version 4.16; National Institutes of Health, Bethesda, MD, USA).

Cells were allowed to differentiate for 6 days at 37°C, followed by incubation at 37°C for 24 h in either SFM (control group) or SFM containing 100 µM DEX (DEX group), 100 µM DEX + 500 ng/ml PYP1-5 (DEX+PYP1-5 group) or 500 ng/ml PYP1-5 (PYP1-5 group). Cells were washed in cold phosphate-buffered saline (PBS) and lysed with extraction buffer [1% NP-40, 0.25% sodium deoxycholate, 1 mM ethylene glycol-bis (β-aminoethyl ether)-N, N,N', N'-tetraacetic acid, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5] containing protease inhibitors (1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin A, 200 mM Na₃VO₄, 500 mM NaF and 100 mM phenylmethylsulfonyl fluoride) on ice. Extracts were centrifuged at 18,341 × g for 10 min at 4°C, and protein levels were quantified using a Bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The supernatant was then used in western blotting.

Cell were treated and harvested as aforementioned, lysed with hypotonic lysis buffer [25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES; pH 7.5), 5 mM EDTA, 5 mM MgCl₂ and 5 mM dithiothreitol (DTT)], and incubated for 15 min on ice. NP-40 (2.5%) was added and the cells were lysed for an additional 10 min. Nuclei were collected by centrifugation at 7,310 × g for 15 min at 4°C. Nuclear proteins were resuspended in extraction buffer (10 mM HEPES pH 7.9, 100 mM NaCl, 1.5 mM MgCl₂, 0.1 mM EDTA and 0.2 mM DTT) and incubated for 20 min at 4°C. Extracts were centrifuged at 18,341 × g for 10 min, and the protein levels were determined using a Bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The supernatant was used then for western blot analysis.

Proteins (20 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked at room temperature for 1 h with 1% bovine serum albumin (BSA) in TBS-T (10 mM Tris-HCl, 150 mM NaCl and 0.1% Tween-20) and then incubated with primary antibodies against Myogenin, MuRF1, MAFbx and GAPDH (Table I; all purchased from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature for 3 h. Membranes were incubated with secondary horseradish peroxidase-conjugated rabbit anti-goat IgG, goat anti-mouse IgG (both Santa Cruz Biotechnology, Inc.) and goat anti-rabbit IgG (Cell Signaling Technology, Inc., Danvers, MA, USA; Table II) at room temperature for 1 h and bands were detected using an enhanced chemiluminescence western blotting kit (Thermo Fisher Scientific, Inc.). Experiments were performed in triplicate and densitometry analysis was performed using Multi-Gauge software version 3.0 (Fujifilm Life Science, Tokyo, Japan).

Total RNA was extracted from C2C12 cells (4×10⁴ cells/well) using TRIzol Reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RNA quality was evaluated by measuring absorbance at 260 and 280 nm to calculate concentration and to assess RNA purity. The RevoScript Reverse Transcriptase PreMix (oligo dT) kit (Intron Biotechnology, Inc., Seongnam, Korea) was used to prepare cDNA, according to the manufacturer's protocol, and the samples were stored at −50°C. qPCR was conducted in 20 µl reactions using the QuantiMix SYBR kit (PhilKorea Technology, Inc., Daejeon, Korea) and the Eco Real-Time PCR System (Illumina, Inc., San Diego, CA, USA), following the manufacturer's protocol. Oligonucleotide primers for MuRF1, atrogin1/MAFbx and GAPDH are shown in Table III. qPCR reactions were incubated for an initial denaturation at 95°C for 10 min, followed by 40 cycles of: 95°C for 15 sec, 55°C for 15 sec, and 72°C for 15 sec. For each sample, the expression level of target mRNA was quantified to those of GAPDH mRNA and calculated using the comparative ΔΔCq method (19). All reactions were performed in triplicate and repeated in two independent experiments.

Multiple mean values were assessed by analysis of variance using SPSS version 10.0 software (SPSS Inc., Chicago, IL, USA) using one-way analysis of variance followed by a Duncan's multiple range test. P<0.05 was considered to indicate a statistically significant difference.

C2C12 skeletal muscle myoblasts were induced to differentiate in a mitogen-poor media such as 2% FBS, fusing to form multinucleate myotubes (20). Fig. 2 illustrates that the cell diameter remained similar following 4, 5 and 6 days of differentiation. In addition, the level of myogenin expression, a factor that regulates terminal differentiation of muscle cells, was similar following 4 and 5 days then increased following 6 days. These results indicate complete differentiation of myoblasts into myotubes at this time (6 days).

MuRF1 and atrogin1/MAFbx protein expression levels were measured in C2C12 myotubes treated with 1, 10, 50 or 100 µM DEX for 24 h (Fig. 3A and B). Treatment with DEX led to increased protein expression levels for MuRF1 and atrogin1/MAFbx, and the levels of expression increased in a dose-dependent manner. These data suggest that DEX may induce muscle atrophy in C2C12 myotubes. In addition, as shown in Fig. 3C, when compared with the control group, the C2C12 myotube was markedly atrophied in the 100 µM DEX-treated group. Therefore, all further experiments were performed following treatment with 100 µM DEX.

Through mass spectrometry, we identified a 1,622 Da compound from P. yezoensis, which we named PYP1-5 (Fig. 1). The MTS assay was used to determine PYP1-5 cytotoxicity to C2C12 myoblasts, which demonstrated no cytotoxic effects following PYP1-5 treatment for 24 h (Fig. 4). Therefore, all further experiments were performed 24 h following treatment with 500 ng/ml PYP1-5, which is the optimum concentration without toxicity as described by Choi et al (18).

C2C12 myotubes were allowed to differentiate for 6 days, followed by 100 µM DEX and/or 500 ng/ml PYP1-5 treatment for a further 24 h. As a result, when compared with the control group, the DEX-treated group exhibited a 36% reduction in cell diameter, whereas the PYP1-5-treated group exhibited a 10% increase in cell diameter. In addition, DEX-induced reduction of cell diameter was suppressed by co-treatment with PYP1-5 (Fig. 5A and B). The anti-atrophic effects of PYP1-5 were investigated by examining the protein and mRNA expression levels of the muscle atrophy markers MuRF1 and atrogin1/MAFbx in C2C12 myotubes that were treated with 100 µM DEX and 500 ng/ml PYP1-5 for 24 h (Fig. 6). Cells treated with DEX alone exhibited a marked increase in the mRNA and protein expression levels of MuRF1 and atrogin1/MAFbx compared with cells in the untreated control group (Fig. 6). The DEX-induced upregulation of MuRF1 and atrogin1/MAFbx was suppressed by co-treatment with 500 ng/ml PYP1-5. Therefore, PYP1-5 may be able to inhibit atrophy and induce hypertrophy pathways in C2C12 myotubes.