C-C motif chemokine ligand 2 promotes myogenesis of myoblasts via the AKT-mTOR pathway

C-C motif chemokine ligand 2 (CCL2) affects cell viability, proliferation, migration, and myogenesis in mouse C2C12 myoblast (MB) and differentiation in myotube (MT)

MBs viability was determined using the Cell Counting Kit-8 (CCK-8) assay (DOJINDO, Kumamoto, Japan). And MBs proliferation was confirmed with the 5-bromo-2′-deoxy-uridine (BrdU) assay (Roche, Germany), relative expression of PCNA (NM_011045.2), and Cyclin D1 (BC044841.1). Within the maximum CCL2 concentration of 800 ng/ml, the greatest increase in viability and proliferation was observed at 200 ng/ml of CCL2 compared with CCL2 vehicle only (21.4 % increase, P<0.0001) (Figure 1A). The relative expression of PCNA and Cyclin D1 increased in a concentration-dependent manner in the CCL2 treatment group compared to the CCL2 vehicle only, and the values were the highest at 200 ng/ml of CCL2 (20.6 % increase, P<0.0001 and 28.0% increase, P<0.001, respectively) (Supplementary Figure 1). Using the concentrations mentioned in other papers [14, 15], 0, 100, and 200 ng/ml were selected based on our CCK-8 and BrdU assay experiments. To investigate cell migration capacity, we conducted a Boyden chamber migration assay. CCL2 increased the migration capacity of MBs compared with CCL2 vehicle only (84.9 % increase, P<0.0005) (Figure 1B).

Figure 1. C-C motif chemokine ligand 2 (CCL2) promotes cell viability, proliferation, migration, and myogenesis in mouse C2C12 myoblasts (MBs). (A) MBs viability on Cell Counting Kit-8 (CCK-8) assay and MBs proliferation on 5-bromo-2′-deoxy-uridine (BrdU) assay. (B) MBs migration on Transwell migration assay. (C) MBs were differentiated into myotubes (MTs) with 2% horse serum after exposure to the indicated concentrations of recombinant CCL2 for the specified number of days. MTs were stained with anti-myosin heavy chain (MyHC) antibody, and the nuclei were counterstained with 4, 6-diamidino-2-phenyindole (DAPI). Scale bars, 100 μm. (D) Quantitative results per field are presented. Number of experiments (No.) of (A, D) = 4 times, respectively. No. of (B, C) = 6 times, respectively. Data are expressed as mean ± standard deviation (SD). (A, B) *P<0.05, **P<0.005, ***P<0.0005, †P<0.01, ††P<0.001, †††P<0.0001 vs. CCL2 '0' (Vehicle: PBS + 0.1% BSA). (C, D) *P<0.05, **P<0.005, ***P<0.0005, †P<0.01, ††P<0.001, †††P<0.0001 vs. Day 1 of CCL2 '0' (vehicle: PBS + 0.1% BSA), or vs. same day data (represented with lines).

MBs were differentiated into MTs in 2% horse serum after exposure to the indicated concentrations of recombinant CCL2 each day. MTs were stained with anti-myosin heavy chain (MyHC) antibody, and the nuclei were counterstained with 4,6-diamidino-2-phenyindole (DAPI). MTs lengthened as CCL2 concentration increased and differentiation time was extended, allowing us to surmise that MTs differentiation was progressing (Figure 1C).

Next, we examined MTs differentiation parameters with CCL2 in immunocytochemistry staining (ICC) data. We set 640 x 540 um² as one field and analyzed 6 to 8 fields for each condition. If there was one nucleus, a cell was considered a myocyte, and two nuclei, a myotube. The fusion index increased with CCL2 exposure compared to 5day CCL2 vehicle only (27.0 % increase, P<0.05). MTs length also increased significantly compared to 5day CCL2 vehicle only (13.1 % increase, P<0.0001) from day 1 to day 7. The number of MTs and the number of mature MTs with three or more nuclei increased in the same pattern. Also, as MTs fusion progressed from day 1 to day 7 of differentiation, the number of myocytes gradually decreased. MTs elongation increased significantly, but MTs width did not show a distinct difference through day 5 (Figure 1D). Collectively, these data indicate that CCL2 contributes to myogenesis by stimulating viability, proliferation, migration, and differentiation.

CCL2 affects cell viability, proliferation, and myogenesis in primary human cells, HSMM (human skeletal muscle myoblast) and differentiation in MT

To validate the mouse cell line studies, HSMM viability was determined using the CCK-8 assay, and HSMM proliferation was confirmed with the BrdU assay, as with the experiments with C2C12. Within the maximum recombinant human CCL2 (hCCL2) concentration of 400 ng/ml, the most significant increase in viability and proliferation was observed at 200 ng/ml of hCCL2 compared with hCCL2 vehicle only (26.7 % increase, P<0.0001) (Figure 2A).

Figure 2. Recombinant human C-C motif chemokine ligand 2 (hCCL2) promotes cell viability, proliferation, and myogenesis in human skeletal muscle myoblast (HSMM) primary human cells. (A) HSMM cell viability on Cell Counting Kit-8 (CCK-8) assay and proliferation on 5-bromo-2′-deoxy-uridine (BrdU) assay. (B) Quantitative reverse-transcription polymerase chain reaction analyses (qRT-PCR) of myogenin and myosin heavy chain (MyHC) as a marker for myotube (MT) formation. (C) MTs were stained with an anti-MyHC antibody, and the nuclei were counterstained with 4, 6-diamidino-2-phenyindole (DAPI). Scale bars, 100 μm. (D) Quantitative results per field are presented. The number of experiments (No.) of (A, B) = 4 times, respectively. No. of (C, D) = 6 times, respectively. Data are expressed as mean ± standard deviation (SD). (A, B) *P<0.05, **P<0.005, ***P<0.0005, †P<0.01, ††P<0.001, †††P<0.0001 vs. hCCL2 '0' (Vehicle: PBS + 0.1% BSA). (C, D) *P<0.05, **P<0.005, ***P<0.0005, †P<0.01, ††P<0.001, †††P<0.0001 vs. Day 5 of hCCL2 '0' (vehicle: PBS + 0.1% BSA).

To investigate how hCCL2 affects the RNA expression of MTs markers, hCCL2 and vehicle (phosphate-buffered saline [PBS] and 0.1% bovine serum albumin solution [BSA]) were administered to MTs. HSMM were differentiated into MTs in 2% horse serum after exposure to the indicated concentrations of hCCL2 each day. Quantitative reverse-transcription polymerase chain reaction analyses (qRT-PCR) at five days showed that myogenin increased dependent on hCCL2 concentration (39.0 % increase, P<0.0001). MyHC increased hCCL2 concentration-dependent manner compared to a 5day CCL2 vehicle only (45.7 % increase, P<0.0001) (Figure 2B).

Next, MTs were stained with MyHC antibody, and the nuclei were counterstained with DAPI. MTs lengthened as hCCL2 concentration increased, allowing us to presume that MTs differentiation was progressing (Figure 2C). We examined MTs differentiation parameters with hCCL2 in ICC data. The fusion index increased with hCCL2 exposure compared to hCCL2 vehicle only (14.2 % increase, P<0.0001). MTs length increased significantly compared to hCCL2 vehicle only (65.3 % increase, P<0.0001). The number of MTs and the number of mature MTs with three or more nuclei increased in the same pattern (97.7 % increase, P<0.0001 and 277.5% increase, P<0.0001, respectively). As MTs fusion progressed, the number of myocytes gradually decreased (49.2 % decrease, P<0.0001). Also, MTs width increased significantly (6.9 % decrease, P<0.005) (Figure 2D). These data indicate that hCCL2 contributes to myogenesis by stimulating viability, proliferation, and differentiation.

Transcription factor expression during myogenesis with CCL2

To investigate how CCL2 affects the RNA expression of MTs markers, qRT-PCR was performed. MRF4 tended to increase, dependent on time and CCL2 concentration (Figure 3A). MyoD increased for three days, until the MTs matured, and then decreased after that. Myogenin increased for three days and decreased after five days. These findings can be visualized because the number of committed myocytes also decreased from day 5. MyHC, a late-stage marker of fusion that reflects the change from committed myocytes to mature MTs, increased in a differentiation-day- and CCL2 concentration–dependent manner compared to 7day CCL2 vehicle only (22.8 % increase, P<0.005) (Figure 3A). Western blot analyses of myogenin and MyHC show that same pattern (Figure 3B). To determine protein synthesis activity during myogenesis after CCL2 treatment, the SUnSET assay was performed. Western blot analysis to quantify relative puromycin incorporation by CCL2 showed that CCL2 increased protein synthesis during myogenesis compared to 3day CCL2 vehicle only (13.4 % increase, P<0.05) (Supplementary Figure 2). All data regarding the RNA expression of transcription factors during MTs differentiation indicated that CCL2 enhances MTs formation, differentiation, and fusion.

Figure 3. C-C motif chemokine ligand 2 (CCL2) effects on transcription factors during myogenesis. (A) Quantitative reverse-transcription polymerase chain reaction analyses (qRT-PCR) of MRF4, MyoD, myogenin, and myosin heavy chain (MyHC) as a marker for myotube (MT) formation. (B) Western blot of MyHC and myogenin in C2C12 cells with 2% horse serum in the presence of the indicated concentrations of recombinant CCL2 for the specified number of days. Number of experiments of (A, B) = 4 times, respectively. Data are expressed as mean ± standard deviation (SD). (A, B) *P<0.05, **P<0.005, ***P<0.0005, †P<0.01, ††P<0.001, †††P<0.0001 vs. day 1 of CCL2 '0' (vehicle: PBS + 0.1% BSA), or vs. same day data (represented with lines).

CCL2 receptor in muscle cells

Among the potential receptors that could mediate the action of CCL2 [18], CC chemokine receptor 2 (CCR2) (not CCR4) was strongly expressed in both MBs and MTs (Figure 4A). In line with those results, CCL2 interacted directly with CCR2 in muscle cells. qRT-PCR indicated that CCR2-siRNA attenuated recombinant CCL2–induced myogenesis compared to siRNA control MTs & CCL2 (42.7 % reduction, P<0.001) (Figure 4B). Western blot analyses of transcription factor expression showed that same pattern compared to siRNA control MTs & CCL2 (47.0 % reduction, P<0.05) (Figure 4C). MTs differentiation parameters in the ICC data revealed that CCR2-siRNA decreased MTs differentiation (Figure 4D). Quantitative results per field also indicated that the presence of CCR2-siRNA attenuated CCL2-induced effects (Figure 4E). These findings suggest that CCR2 mediates the effects of CCL2 on myogenic differentiation. The CCL2 concentration used here was 200 ng/ml.

Figure 4. C-C chemokine receptor 2 (CCR2) is the major receptor for C-C motif chemokine ligand 2 (CCL2) in muscle cells. Mouse C2C12 myoblasts (MBs) were differentiated into myotubes (MTs) with 2% horse serum for the indicated number of days. (A) Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blot analyses were performed to determine the expression of CCR2 in C2C12 cells cultured with 2% horse serum for the specified number of days. (B) Mouse C2C12 MBs were differentiated into MTs with 2% horse serum in the presence or absence of recombinant CCL2 and/or CCR2 inhibitor for three days. qRT-PCR results for CCR2, MyoD, MyoG, and myosin heavy chain (MyHC) in C2C12 cells. (C) Western blots of CCR2, MyHC, and myogenin in C2C12 cells cultured with 2% horse serum in the presence or absence of recombinant CCL2 and/or CCR2 inhibitor for three days. (D) MTs were stained with anti-MyHC antibody, and the nuclei were counterstained with 4, 6-diamidino-2-phenyindole (DAPI). Scale bars, 100 μm. (E) Quantitative results per field are presented. Number of experiments (No.) of (A–C) = 4 times, respectively. No. of (D, E) = 6 times, respectively. Data are expressed as mean ± standard deviation (SD). (A) *P<0.05, **P<0.005, ***P<0.0005, †P<0.01, ††P<0.001, †††P<0.0001 vs. MB. (B, C) *P<0.05, **P<0.005, ***P<0.0005, †P<0.01, ††P<0.001, †††P<0.0001 vs. siRNA Control MB, or vs. siRNA Control MT or siRNA Control MT & CCL2 (represented with lines). (E) *P<0.05, **P<0.005, ***P<0.0005, †P<0.01, ††P<0.001, †††P<0.0001 vs. siRNA Control MT, or vs. siRNA Control MT & CCL2 (represented with lines).

CCL2 effects on MTs damaged by dexamethasone

To confirm the importance of CCL2 in protection against myotube atrophy, we induced MTs atrophy with dexamethasone [19, 20] which impairs RNA expression of the transcription factors needed for MTs differentiation. We found that CCL2 restored the damage to MTs caused by dexamethasone (21.8 % increase, P<0.0001) (Figure 5A). The ICC data also show that MTs differentiation was impaired by dexamethasone, and that CCL2 restored myotube atrophy (Figure 5B, 5C). Treating MTs with CCL2 significantly reversed dexamethasone-mediated MTs atrophy.

Figure 5. C-C motif chemokine ligand 2 (CCL2) restores myotubes (MTs) damaged by dexamethasone. (A) Quantitative reverse-transcription polymerase chain reaction analyses (qRT-PCR) of myogenin and myosin heavy chain (MyHC) in C2C12 cells treated with 2% horse serum, with and without MTs damaged by dexamethasone or the indicated concentrations of recombinant CCL2 for 2 days. (B) MTs were stained with anti-MyHC antibody, and the nuclei were counterstained with 4,6-diamidino-2-phenyindole (DAPI). Scale bars, 100 μm. (C) Quantitative results per field are presented. Number of experiments (No.) of (A) = 4 times. No. of (B, C) = 6 times, respectively. Data are expressed as mean ± standard deviation (SD). (A, C) *P<0.05, **P<0.005, ***P<0.0005, †P<0.01, ††P<0.001, †††P<0.0001 vs. CCL2 '–' & Dexa ‘– ‘(vehicle: PBS + 0.1% BSA), or vs. CCL2 '–' & Dexa 200μM (represented with lines).

Identification of the signal-mediating role of CCL2 during myogenesis

To determine the mechanism by which CCL2 affects myogenesis, we focused on the AKT-mTOR signaling pathways, which are related to protein synthesis in myogenesis [21]. Western blot analyses revealed that CCR2 expression increased with CCL2 administration and decreased with CCR2-siRNA exposure (Figure 6A, 6B, respectively). The activity of p-mTOR/mTOR and p-AKT/total AKT also increased with CCL2 compared to CCL2 vehicle only (18.3 and 30.5% increase respectively, P<0.05) and decreased with CCR2-siRNA compared to CCL2 (38.9 % (P<0.05) and 56.7% (P<0.005) reduction respectively) (Figure 6C, 6D, respectively). These results indicate that AKT-mTOR is a crucial signal regulating the effects of CCL2 on myogenesis. To detect whether the effects of CCL2 promoting myogenesis can be blocked by the inhibitor, we conducted Western blot analyses with AKT-mTOR inhibitor (Supplementary Figure 3A). The activity of p-mTOR/mTOR and p-AKT/total AKT decreased with Akt inhibitor compared to CCL2 (56.2% (P<0.005) and 94.3% (P<0.005) reduction respectively) (Supplementary Figure 3B). And also, the relative intensity of MyHC and myogenin decreased with Akt inhibitor compared to CCL2 (60.5% (P<0.05) and 67.8% (P<0.01) reduction, respectively) (Supplementary Figure 3C).

Figure 6. Effects of C-C motif chemokine ligand 2 (CCL2) on myogenesis are mediated by AKT-mTOR signal stimulation. (A, B) Western blot analyses to determine the activity of signals related to myogenesis in C2C12 cells after treatment with 200 ng/ml CCL2. (C, D) Quantitative reverse-transcription polymerase chain reaction analyses (qRT-PCR) of the relative intensity of p-mTOR/mTOR or p-AKT/total AKT in C2C12 cells treated with 2% horse serum in the presence or absence of recombinant CCL2 and/or CCR2 inhibitor for three days. Number of experiments (No.) of (A–D) = 4 times, respectively. Data are expressed as mean ± standard deviation (SD). (B–D) *P<0.05, **P<0.005, ***P<0.0005, †P<0.01, ††P<0.001, †††P<0.0001 vs. siRNA Control MB, or vs. siRNA control MT, or vs. siRNA control MT & CCL2 (represented with lines).

Cell culture and reagents

Mouse C2C12 cells were maintained in growth medium (DMEM, Welgene, Gyeongsan-si, Korea) containing 10% fetal bovine serum (FBS; Gibco-BRL, Waltham, MA, USA), 1% v/v penicillin–streptomycin (10,000 U/mL of penicillin and 10,000 g/mL of streptomycin, Gibco-BRL) at 37° C in a humidified atmosphere containing 5% CO2. Differentiation was induced by switching to a low serum medium (DMEM + 2% horse serum (Gibco-BRL), 1% v/v penicillin–streptomycin). The medium was replaced every day.

HSMM (Lonza, CC-2580, Walkersville, MD, USA) were cultured in skeletal muscle growth media-2 (Lonza, CC-3246) supplemented with SingleQuots™ Kit (human epidermal growth factor, Dexamethasone, L-glutamine, FBS, and Gentamicin/ Amphotericin-B; Lonza, CC-3244) and incubated under 5% CO2 at 37° C. To study MTs, after 1–2 days (when the cells reach 80–90% of confluence) of seeding with growth media, the cells were cultured in a differentiation media consisting of DMEM: F-12 (Lonza, 12-719F, Walkersville, MD, USA) supplemented with 2% horse serum. During the complete differentiation process (5 days), cells were exposed to hCCL2 (R&D Systems, Minneapolis, MN, USA) or vehicle control (0.1% BSA in PBS) and incubated in the same differentiation media. The differentiation media was changed every 48 hours with hCCL2 or vehicle control. For the dexamethasone test, after MTs were fully differentiated for 3-4 days, the cells were cotreated with CCL2 or hCCL2 (100, 200 ng/ml) and dexamethasone 200 μm for 24 h. Recombinant mouse CCL2/JE/MCP-1 and hCCL2 were purchased from R&D Systems (Minneapolis, MN, USA). Dexamethasone was purchased from Sigma Aldrich (St. Louis, MO, USA).

Cell viability assay

Cell viability was measured using a CCK-8 assay (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. MBs cells (1×10⁴ cells/well, 100 μl) were seeded into 96-well plates for 24 h. Fresh serum free medium containing the indicated concentrations of CCL2 or hCCL2 was added. The control ‘0’ was treated with vehicle of CCL2 or hCCL2 only (0.1% BSA in PBS). After incubation for 24 h, culture medium was replaced with drug-free medium (100 μl). Briefly, 10 μl of WST-8 dye (2-[2-methoxy-4-nitrophenyl]-3-[4-nitrophenyl]-5-[2, 4-disulfophenyl]-2H-tetrazolium, monosodium salt) was added to each well of a 96-well plate [47]. The suspension was incubated for 2 h, and the absorbance was read at 450 nm, with a reference wavelength of 650 nm, using a microplate reader (BioTek Epoch Gen5, Houston, TX, USA).

Proliferation assay

C2C12 MBs (1×10⁴ cells/well, 100μl) were seeded into 96-well plates for 24 hours. Fresh serum free medium containing the indicated concentrations of CCL2 was added. Cell proliferation was measured by monitoring BrdU incorporation. Cells were incubated with BrdU for 3 hours, and then cell proliferation was assayed using a BrdU labeling and detection kit (Roche, Mannheim, Germany). Absorbance was measured at 450 nm, with a reference wavelength of 650 nm, using a microplate reader (BioTek Epoch Gen5).

Migration assay

To examine cell migration capacity, we used a Boyden chamber system with Transwell plates and 8-μm-porosity polycarbonate membranes (Corning, NY, USA). Cells were seeded onto the inner chamber at a density of 1.0 × 10⁵ cells/200 μL in DMEM with 0.1% FBS and then exposed to CCL2 (100 and 200 ng/ml) in the outer chamber for 24 hours [48]. Cells on the upper membrane were removed by wiping with a cotton swab, whereas cells on the lower surface of the membrane were fixed in 4% paraformaldehyde (PFA) for 2 min and stained with 0.05% crystal violet. Images of the lower side of the membrane were produced using a microscope (Olympus CKX-41, Tokyo, Japan). The total number of migrated cells for each well was quantified using ImageJ software.

Quantitative reverse-transcription PCR (qRT-PCR)

Total RNA was extracted from C2C12 or HSMM cells with TRIzol reagent (Sigma Aldrich, St. Louis, MO, USA). We used RNA to cDNA EcoDry Premix (Takara, Shiga, Japan) to carry out cDNA synthesis for mRNA. The analysis of mRNA expression was carried out with Faststart Essential DNA Green Master (Roche, Mannheim, Germany) in a Roche Light Cycler 96 system (Roche, Mannheim, Germany) following the manufacturers’ instructions. Primers for anti-MyHC (NM_001039545.2, BC093082.1), myogenin (NM_031189.2, NM_002479.6), MyoD (NM_010866.2), Myf5 (NM_008656.5), MRF4 (NM_008657.2), PCNA (NM_011045.2), Cyclin D1 (BC044841.1), and CCR2 (NM_009915.2) were obtained from Applied Biosystems (Foster City, CA, USA). The threshold cycle (Ct) value for each gene was normalized to the Ct value of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (M_008084.3, BC083511.1).

Western blot analysis

C2C12 cells were lysed at 4° C in RIPA lysis buffer (Enzo Life Sciences, Farmingdale, NY, USA), and the lysate was centrifuged at 13,000 rpm and 4° C for 20 min. Aliquots of the supernatant were removed for protein analysis by the Bicinchoninic acid (BCA) protein assay method (Pierce, Rockford, IL, USA). The supernatant was incubated at 95° C for 5 min in 4x Laemmli Sample Buffer (Bio-Rad, Laboratories, Hercules, CA, USA) containing 355mM 2-mercaptoethanol, and 20μg of the sample was resolved by 10% SDS-PAGE. The separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Molsheim, France). The primary antibodies [phospho-mTOR (Ser2448), mTOR, phospho-Akt (Ser473), and Akt] and HRP-conjugated secondary antibodies (HRP-linked anti-mouse IgG and anti-rabbit IgG) were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibodies against anti-MyHC and anti-α-tubulin were purchased from Sigma Aldrich. Myogenin and CCR2 primary antibodies were purchased from Abcam (Cambridge, UK). Immunoreactive bands were visualized using a LAS-AI600 enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Arlington Heights, IL, USA). Protein expression levels were normalized to that of α-tubulin, and the western blotting band signal intensities were quantified using ImageJ software.

Immunofluorescence

C2C12 or HSMM cells were grown on coverslips (SPL Life sciences, Phocheon-si, Korea, Cat#20018). To evaluate the protective effects of CCL2 or hCCL2 on dexamethasone-induced muscle atrophy, differentiated MTs were cotreated with recombinant CCL2 or hCCL2 (100, 200 ng/ml) and dexamethasone (200 μm) for 24 hours. C2C12 or HSMM MBs and MTs were fixed in ice-cold 4% PFA for 15 min, washed three times with PBS, and incubated in ice-cold 0.25% Triton X-100 at room temperature for 10 min [49]. Then, cells were blocked with blocking solution (1% BSA in PBS) and washed three more times. Next, cells were incubated overnight in anti-MyHC (1:400, Sigma Aldrich, St. Louis, MO, USA) at 4° C. Following three washes in PBS, the cells were incubated with Alexa 555–labeled anti-mouse IgG antibodies (1:1000, Cell Signaling, MA, USA) for 60 min. Then, cells were stained with 4, 6-diamidino-2-phenyindole (DAPI, 1:5000, Sigma Aldrich, St. Louis, MO, USA) for 10 min and washed with PBS three times. The images were produced using an LMS800 fluorescence microscope (Carl Zeiss, Oberkochen, Germany). The fusion index (%) was calculated using the following equation: 100×number of nuclei in MyHC+ MTs per total number of nuclei in MyHC+ myocytes and MTs.

siRNA transfection

C2C12 MB cells (1.5×10⁵ cells/well) were seeded into 6-well plates for 24 h. CCR2 siRNA oligonucleotides (NM_009915.2), CCR4 siRNA oligonucleotides (NM_009916.2), and negative control siRNA oligonucleotides were synthesized by Bioneer Corp. AccuTarget™ negative control siRNA (SN-1001) was also purchased from Bioneer (Daejeon, Korea). siRNA oligonucleotides were transfected into C2C12 cells using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA) as advised by the manufacturer’s protocol. The siRNA concentration used was 100nM. Briefly, each siRNA and lipofectamine 3000 (Invitrogen) was diluted in serum-free OPTI-MEM and incubated for 5 min, respectively. The diluted siRNA and lipofectamine 3000 reagent were mixed by inverting and incubated for 20 min to form complexes. Pre-formed complexes were added directly to the cells and cells were incubated for an additional 6 h. Then, culture medium was replaced with antibiotic and 10% FBS-containing DMEM and incubated. The next day, differentiation media and CCL2 200 ng/ml were administered together. We used MTs from day 3 of differentiation for our experiments.

MK2206-mediated Akt inhibition

C2C12 cells were treated with 5 μM specific Akt inhibitor MK2206 (MedChemExpress, HY-10358, Monmouth Junction, NJ, USA) and 200 ng/ml CCL2 for three days of differentiation. Re-adding MK2206, CCL2, or vehicle control (DMSO) changed daily.

SUnSET assay by Western blotting

To measure in vitro protein synthesis using the SUnSET assay, C2C12 cells were treated with 1 μM puromycin (P8833, Sigma Aldrich) for 30 min before harvesting. Cell lysates were normalized for equal amounts of protein using the BCA Protein assay method (Pierce). Samples were boiled with 4x Laemmli Sample Buffer (Bio-Rad) containing 355 mM 2-mercaptoethanol, and 10 μg of the sample was resolved by 10% SDS-PAGE. The proteins were transferred onto a 0.45 μm PVDF membrane (Merck Millipore). Blocking was conducted for 30 min with 3% skim milk prepared in Tris-buffered saline with 0.05% Tween 20 (TBST). Blots were washed thrice in TBST and incubated with anti-puromycin (1:25000, Millipore, MA, USA) as the primary antibody. Membranes were washed thrice with TBST and incubated with HRP-conjugated anti-mouse IgG (Cell Signaling Technology, Danvers, MA, USA) as the secondary antibody. Immunoreactive bands were visualized using a LAS-AI600 enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Arlington Heights, IL, USA). Protein expression levels were normalized to that of α-tubulin, and western blotting band signal intensities were quantified using ImageJ software.

Statistical analysis

The data are expressed as mean ± standard deviation (SD) from at least three independent experiments relying on triplicate measurements, unless otherwise specified. We checked the normality using the Shapiro-Wilk test. For items that satisfied normality, Student’s t test and analysis of variance (ANOVA) with post hoc analysis via Tukey’s honest significance test were used to evaluate the significance of differences between two groups and among ≥3 groups, respectively. For items that did not satisfy normality, the Mann-Whitney U test and the Kruskal Wallis test were used to evaluate the significance of differences between two groups and among ≥3 groups, respectively. P values <0.05 were considered statistically significant. SPSS version 20.0 (SPSS, Inc., Chicago, IL, USA) was used for statistical analyses.