All animal handling procedures were approved by the Institutional Animal Care Committee of Taipei Medical University. The animal living conditions were in accordance with the standards of the Guide for the Care and Use of Laboratory Animals (2011), eighth edition, published by National Research Council and the Guidelines of the Animal Research Committee of Taipei Medical University.
Isolation of myogenic cells from TA muscle
A procedure described in a previous study  was used for isolation of myogenic cells. Briefly, Sprague Dawley rats aged 6–8 weeks and weighing 190–210 g were used in this experiment. After scarification, the TA muscle was excised and homogenized with scissors. The muscle tissue was digested for 30 min at 37℃ by using 0.5% trypsin-EDTA (Thermo Fisher Scientific, Waltham, MA, USA). The sample was collected through centrifugation at 1500 × g for 5 min and titrated through a fine pipette. An enriched population of myogenic cells was recovered by differential centrifugation (500 × g for 1 min followed by 1500 × g for 5 min). The pellet was suspended in Dulbecco’s Modified Eagle Medium (DMEM) containing 5% horse serum (Sigma-Aldrich, St. Louis, MO, USA). The suspension containing myogenic cells was then filtrated though a nylon membrane (200 μm) and plated onto 0.1% gelatin coated petri dish. The isolated cells were cultured in growth medium that comprised DMEM with 5% horse serum and 10% fetal bovine serum and 2 mg/mL, 200 μg/mL and 20 μg/mL of mSC, under 5% CO2 at 37℃. The cell number was counted after 2 days and 8 days of plating for proliferation evaluation until it was confluent for myofiber quantity evaluation.
Please see supplementary materials for details.
Physical analysis and rheological measurement of mSC
Please see supplementary materials for details.
Immunohistochemical staining and BrdU assay
The procedure of immunohistochemical analysis was performed as described in a previous study [30, 31]. Briefly, the cultured cells were fixed in 2% paraformaldehyde. Mouse anti-Pax7 (MAB1675; R&D Systems, Minneapolis, MN, USA) at a dilution of 1:50 and anti-Pax7-Cf750 conjugate (Pax7/CF750; 92284, Biotium, Fremont, CA, USA) antibodies were used for Pax7 staining. Rabbit polyclonal anti-BrdU antibody (ab152095; Abcam, Cambridge, UK) was used according to the manufacturer instructions. In addition, rabbit anti-Toll-like receptor 2 (TLR2, ab191458; Abcam, Cambridge, UK) and mouse anti-Toll-like receptor 4 (TLR4, ab22048; Abcam, Cambridge, UK) antibodies were used as primary antibodies. Regarding secondary antibodies, the FITC-labeled anti-mouse IgG, Cy3-labeled anti-rabbit IgG, FITC-labeled anti-rabbit IgG, and Cy3-labeled anti-mouse IgG (715-095-151, 711-165-152, 711-085-152, 715-165-151; Jackson ImmunoResearch Laboratory, West Grove, PA, USA) were used for Pax7, BrdU, TLR2, and TLR4 detection, respectively. Hoechst 33324 (H3570; Invitrogen, Paisley, UK) and DAPI (Biotium 40011, Fremont, CA, USA) were used for nuclear staining. The samples were analyzed through fluorescence microscopy (Olympus, Tokyo, Japan) or on an AS-MDW system (Leica Microsystems, Wetzlar, Germany). Micrographs were obtained using AxioCam (Carl Zeiss Vision, Hallbergmoos, Germany) or the AS-MDW system (Leica Microsystems). The cell counting was performed 48 h after plating. For cell counting analyses, Pax7 positive (Pax7+) and Pax7-BrdU double positive (Pax7+ BrdU+) cells were enumerated per field (×100) in the culture dish, and the average values and their standard deviations (SDs) were calculated from 10 fields for each culture dish sample. The average ratio of Pax7+ BrdU+ cells of each group was acquired using the following formula: (Pax7+ BrdU+) /Pax7+ /control .
The extracts of primary cultures taken at 36 h or 8 days after plating were subjected to standard procedures. The protein amount was determined using BioRad protein assay. Equal amounts of protein (30 µg) were electrophoresed on 10% acrylamide gel and electrotransferred to polyvinylidene fluoride membrane, followed by immunoblotting with antibodies for myosin heavy chain (MyHC; 1:1000, GTX73432, GeneTex, Taiwan), phospho-TAK1 (1:1000, Cell signaling, #4508), p-p38 (1:1000, Cell signaling, #4511), p-JNK (1:1000, GTX52328, GeneTex, Taiwan), p-ERK (1:1000, Cell signaling, #4370), and p-NF-kB (1:1000, Cell signaling, #3033). Goat anti-actin-HRP (1:5000, Santa Cruz Biotechnology) was used for protein quantification. The membrane was washed and incubated with secondary anti-mouse IgG-HRP (1:3000, sc-2005, Santa Cruz, California, USA), anti-goat IgG-HRP (1:3000, sc-2020, Santa Cruz, California, USA), and anti-rabbit IgG-HRP (1:3000, GTX213110-01, GeneTex, Taiwan). Detection was performed using the T-Pro LumiLong Plus Chemiluminescence Detection Kit (JT96-K004M, T-Pro Biotechnology, Taiwan) and the blots were directly processed on an Amersham Imager 600 (GE, Boston, USA). Densitometry was performed using ImageJ.
Signaling pathway inhibition
The inhibition of JNK-MAPK, p38-MAPK, and ERK-MAPK signaling pathways was achieved by addition of SP600125 (s5567, Sigma-Aldrich, St. Louis, MO, USA), SB203580 (s8307, Sigma-Aldrich, St. Louis, MO, USA), and U0126 (19-147, Sigma-Aldrich, St. Louis, MO, USA), respectively. TIRAP (TLR2 and TLR4) inhibitor (tlrl-prslps, Invivogen, San Diego, CA, USA) and TLR2 inhibitor (tlrl-oxpl, Invivogen, San Diego, CA, USA) were also used in this experiment. The all dosages of inhibitors were 1 mg/ml.
AP-1 ELISA assay was performed using AP-1 Activity Assay Kit (GeneCopoeia Inc. MD. USA) according to the protocol provided by the manufacturer . Nuclear fraction (50 mg) was mixed with the transcription factor–binding buffer supplied by the manufacturer and was then added to each well of the 96-well plate coated with oligo-DNA fragment containing consensus AP-1 binding sequence. After incubation for 1 h at room temperature (RT), the wells were washed with 200 mL of washing buffer supplied by manufacturer for 1 min. After the final wash, 100 mL of diluted anti-AP-1 antibody (1:1000) solution was added to each well except the blank wells, and the plate was incubated for 1 h at RT with gentle rocking. Then, each well was washed two more times with washing buffer and incubated with 100 mL of diluted peroxidase-conjugated secondary antibody (1:1000) at RT for 1 h. After another two washes, each well was treated with chemiluminescence developing solution followed by 30 min incubation at RT with gentle agitation and light shielding. After incubation, 100 mL of the stop solution was added to each well and absorbance was measured at 450 nm wavelength using a spectrophotometric plate reader. Nuclear extract of MCF-7 cells was used as a positive control for this assay.
Data are presented as mean ± standard deviation (SD). Statistical analyses regarding different mSC concentration were conducted using one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons comparing the mean of each group to the mean of control. One-way ANOVA with Tukey’s multiple comparisons comparing mean of each groups were conducted for evaluation of SC proliferation with inhibitors. The rest of data were analyzed by unpaired t-test. The significance was set at p ≤ 0.05. Data were analyzed using GraphPad Prism (version 6.0; GraphPad Software, San Diego, CA, USA).