DMEM was purchased from Gibco; Thermo Fisher Scientific, Inc. Penicillin-streptomycin solution and fetal bovine serum (FBS) were purchased from HyClone; GE Healthcare Life Sciences. Trypsin-EDTA (0.05%) was obtained from Gibco; Thermo Fisher Scientific, Inc. Antibodies specific for β-actin (cat. no. sc-47778), GH receptor (GHR; cat. no. sc-57161), IGF-1 receptor β (IGF-1Rβ; cat. no. sc-713), STAT5b (cat. no. sc-1656) and horseradish peroxidase-conjugated goat anti-mouse (cat. no. sc-516102) and anti-rabbit (cat. no. sc-2357) secondary antibodies were obtained from Santa Cruz Biotechnology, Inc. An anti-IGF-1 antibody (cat. no. ab9572) was purchased from Abcam, and pIGF-1Rβ (cat. no. 3021s), pJak2 (cat. no. 3776s), Jak2 (cat. no. 3230s) and pSTAT5 (cat. no. 9314s) antibodies were purchased from Cell Signaling Technology Inc. Recombinant growth hormone (cat. no. 100-40) was purchased from PeproTech Inc. NTS was purchased from Nara Bio Co., Ltd.

Mouse muscle C2C12 cells (ATCC CRL-1772) were cultured in DMEM supplemented with 10% FBS and 1% penicillin and streptomycin at 37°C in 5% CO₂. For each experiment, cells at 70-80% confluence were gently washed twice with PBS. For differentiation studies, cells at 85% confluence were transferred in DMEM supplemented with 2% horse serum (differentiation medium) and treated with NTS in fresh media. Unless otherwise specified, cells were treated with 0.2 µg/ml NTS in 99.9% DMSO (Sigma-Aldrich; Merck KGaA) for 24 h at 37°C.

Cell viability was assessed using an MTT assay (Sigma-Aldrich; Merck KGaA). The day before treatment, cells were re-suspended in DMEM at a density of 1×104 cells/well in 24-well culture plates. The next day, the medium was replaced with fresh DMEM alone (vehicle control) or with different concentrations of NTS (0.1-10 µg/ml), followed by a 24 h incubation at 37°C. Subsequently, MTT (5 mg/ml) was added and the cells were incubated at 37°C for 4 h. The resulting formazan product was dissolved in DMSO, and the absorbance was measured at 560 nm using an Ultra Multifunctional Microplate Reader (Tecan US, Inc.). All measurements were performed in triplicate, and experiments were repeated at least thrice.

Whole cell lysates were prepared from untreated or NTS-treated mouse muscle cells in Radioimmunoprecipitation assay buffer (EMD Millipore) containing phosphatase and protease inhibitors on ice. Cells were disrupted by aspiration through a 23-gauge needle, and the lysates were centrifuged at 18,300 × g and 4°C for 10 min to remove cellular debris. Protein concentrations were measured using the Bradford method (Thermo Fisher Scientific, Inc.). Equal amounts of protein (100 µg/lane) were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked for 1 h with 5% skimmed milk (BD Biosciences) in TBS-T buffer (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1X Tween-20). The membranes were probed overnight at 4°C with relevant primary antibodies diluted in 5% bovine serum albumin (EMD Millipore) or skimmed milk, washed with TBS-T and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies. Visualization was performed using an Enhanced Chemiluminescence Plus detection kit (Amersham; GE Healthcare) and an ImageQuant LAS-4000 imaging device (Fujifilm) with LAS-4000 Control Software (Version 1.1; Fujifilm). The blots were stripped using Restore Western Blot Stripping Buffer (Thermo Fisher Scientific, Inc.).

Total RNA was extracted from cells using the RNeasy Mini kit (Qiagen GmbH) according to the manufacturer's protocol and quantified spectrophotometrically at 260 nm. Subsequently, RT-PCR analyses were performed to detect IGF-1 and GAPDH RNA expression. Briefly, cDNA was synthesized from total RNA at 42°C for 1 h and 95°C for 5 min using a first-strand cDNA synthesis kit (cat. no. K-2041; Bioneer Corporation) and oligo d(T) primers. A RT-PCR Premix kit (cat. no. K-2016; Bioneer Corporation) was used to amplify IGF-1 and GAPDH using specific primers (Bioneer Corporation): IGF-1 forward, 5′-CTA CGC CAA TGT GGT GCT AT-3′ and reverse, 5′-TCT GCC ATT TGC CTG AAG TT-3′ (409 bp); GAPDH forward, 5′-AAG GCC ATC ACC ATCT TCC A-3′ and reverse, 5′-ACG ATG CCA AAG TGG TCA TG-3′ (320 bp). The PCR conditions were as follows: 95°C for 10 min; 32 cycles at 95°C for 45 sec, 58°C for 60 sec and 72°C for 60 sec; and 72°C for 10 min. The PCR products were resolved by electrophoresis on a 2% agarose gel and visualized using ethidium bromide (Sigma-Aldrich; Merck KGaA). Davinch-K Gel Imaging System (Davinch-K Co., Ltd.) and Multi Gauge V3.1 (Fujifilm) were used for densitometry analysis.

C2C12 cells (2×10⁵) were cultured in 6-well plates to 60% confluence. The cells were transfected with a STAT5b-pMX vector (22) (kindly provided by Dr Koichi Ikuta, Kyoto University, Japan) using the DharmaFECT transfection reagent (GE Healthcare Dharmacon, Inc.) for 24 h at 37°C. Transfected cells were washed with ice-cold PBS and treated for 24 h with media containing DMSO with or without NTS. Western blotting was used to isolate and analyze target protein and β-actin expression levels as described above.

C2C12 cells (2×10⁵) were cultured in 6-well plates to 60% confluence and subsequently transfected with On-Target plus SMARTpool siRNA specific for STAT5b or non-targeting siRNA (cat. no. L-010539-00-0005; GE Healthcare Dharmacon) with FuGENE6 (Roche Diagnostics) according to the manufacturer's instructions. After 24 h, NTS was added for another 24 h at 37°C. Proteins were isolated and subjected to western blotting for further analysis as described above.

The ChIP assay was performed using the Imprint^® Chromatin Immunoprecipitation kit (Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. Briefly, mouse muscle cells were fixed with 1% formaldehyde for 10 min at room temperature and quenched with 1.25 M glycine. Following washing with PBS, the cells were suspended in nuclear preparation buffer and shearing buffer and sonicated under optimized conditions (amplitude, 25%; pulse, 30 sec on/30 sec off for 20 min on ice). The sheared DNA was centrifuged at 21,000 × g for 10 min at 4°C, and the cleared supernatant was subjected to protein/DNA immunoprecipitation. The clarified supernatant was diluted with the dilution buffer (ratio, 1:1), and 5 µl aliquots of the diluted samples were used as internal controls. The diluted supernatant was incubated with anti-STAT5b antibodies in Parafilm pre-coated wells for 90 min. The controls were incubated with normal goat IgG and anti-RNA polymerase II. Unbound DNA was removed using immunoprecipitation wash buffer, and bound DNA was collected using the cross-link reversal method with DNA release buffer containing proteinase K. The released DNA and internal control DNA were purified using the GenElute Binding Column G. DNA was then quantified using conventional quantitative PCR. The primer sequences were as follows: IGF-1 forward 5′-CCA CAC ACA CCT ATT CAC CC-3′ and reverse, 5′-CCT GGA GCC ATA GGG TAT GA-3′. The qPCR conditions were as follows: 95°C for 3 min; 40 cycles at 95°C for 30 sec, 60°C for 30 sec and 72°C for 40 sec; and 72°C for 5 min.

Analysis of the growth hormone levels was conducted using Human Growth Hormone SimpleStep^® ELISA kit (ab190811; Abcam). C2C12 cells (2×10⁵) were cultured in 6-well plates to 60% confluence and treated with 99.9% DMSO and NTS for 24 h. The samples were centrifuged at 2,000 × g for 10 min, and the supernatants were collected. The samples were then added to 96-well plate along with equal amounts of antibody cocktail and incubated for 1 h at room temperature on a plate shaker at 400 rpm. The plates were washed with 1X wash buffer, and the development solution was added and incubated for 10 min in the dark on a plate shaker set to 400 rpm. Finally, the stop solution was added, and optical density was recorded at 450 nm using an Ultra Multifunctional Microplate Reader (Tecan US, Inc.). All measurements were performed in triplicate, and experiments were repeated at least thrice.

Data are expressed as the mean ± SEM of at least three experiments. Statistical analysis was performed using Student's t-test or one-way ANOVA with Tukey's post hoc test. Analyses were performed using the SAS 9.3 program (SAS Institute, Inc.). P<0.05 was considered to indicate a statistically significant difference.

NTS is not easily dissolved in common solvents such as water, ethanol or DMSO. Through a series of experiments involving normal and heated water, DMSO and ethanol, NTS was determined to dissolve completely in DMSO at a maximum concentration of 1 mg/ml (Table SI and Fig. S1). To evaluate the effects of NTS on C2C12 cell proliferation, MTT assay was conducted using 0.1-10 µg/ml NTS and DMSO alone (control). NTS induced significantly more cell death compared with an equal amount of DMSO (Fig. 1). For further experiments, NTS concentrations <0.5 µg/ml were selected, since they did not induce significantly greater cell death compared with the controls.

In the MTT assay, NTS concentrations ≤0.5 µg/ml induced <18% mortality in C2C12 mouse muscle cells. Western blotting was used to analyze translational expression of GHR, pSTAT5 and IGF-1 in C2C12 cells treated with 0.05-0.5 µg/ml NTS. The results demonstrated that compared with lower concentrations, 0.2 µg/ml NTS increased the expression of GHR, pIGF-1Rβ, pJak2, pSTAT5 and IGF-1 without inducing notable changes in total Jak2, STAT5b and IGF-1Rβ (Figs. 2A and S3). Slight decreases in the levels of GHR, pSTAT5 and IGF-1 proteins were observed at 0.5 µg/ml NTS, possibly due to increased cell death (Fig. 2B). To confirm the ability of NTS to induce growth hormone signaling, a growth hormone assay was performed in C2C12 cells in presence of NTS, and the results revealed increased levels of growth hormone in NTS compared with control cells (Fig. S2).

As NTS increased the levels of GHR, pSTAT5 and IGF-1, it was hypothesized that it may exert similar effects to those of GH signaling. Western blotting was used to analyze cells treated with 0.1 µg/ml recombinant GH (Fig. 3A). The patterns of GHR, STAT5b, pSTAT5, Jak2, pJak2 and IGF-1 expression were similar between cells treated with NTS and GH (Figs. 3B and S4). pIGF-1Rβ and IGF-1Rβ expression did not exhibit any differences compared with the control. Igf1 mRNA levels in the presence of 0.2 µg/ml NTS or 0.1 µg/ml GH were also evaluated (Fig. 3C). Similar to the protein levels, increased Igf1 mRNA expression was observed in response to NTS and GH, suggesting that these factors induce similar signaling pathways (Fig. 3D).

As NTS and GH signaling exhibited similar effects, the effects of NTS on the binding of STAT5b to the Igf1 promoter region were evaluated using 0.1 µg/ml recombinant GH for comparison. The primer was designed to cover the Igf1 promoter region. The results of ChIP with a STAT5b antibody demonstrated that 0.2 µg/ml NTS increased the binding activity of STAT5b to the Igf1 promoter and thus may have initiated transcription (Fig. 4A). The increased formation of the STAT5b/Igf1 complex in response to GH signaling also suggested similarity with NTS activity (Fig. 4B).

Considering the similarities between NTS activity and GH signaling, the role of STAT5b was compared with NTS treatment by overexpressing STAT5b. Western blotting analysis of NTS-treated and STAT5b-overexpressing cells exhibited similar increases in the levels of GHR, IGF-1 and pSTAT5 (Fig. 5A). NTS also upregulated pJak2, which suggested that it may induce GH signaling by regulating Jak2/STAT5b/IGF-1 signaling. Similar upregulation of GHR in response to NTS treatment and STAT5b overexpression also suggested an association between these signaling axes (Fig. 5B).

To confirm the role of STAT5b in the NTS-mediated regulation of IGF-1, Stat5b expression was silenced using siRNA. Western blotting analysis of on-target STAT5b inhibition revealed increases in the levels of pSTAT5 and IGF-1 following NTS treatment (Fig. 6A). The relative levels of pSTAT5 and IGF-1 suggested that STAT5b may be a key mediator of the effects of NTS in C2C12 mouse muscle cells (Fig. 6B). These results suggested that NTS may act as a GH mimic to regulate STAT5b and IGF-1 signaling.