murine c2c12 skeletal muscle myoblast cells  (ATCC)

Journal: Antioxidants

Article Title: Ginsenoside Rc, an Active Component of Panax ginseng , Alleviates Oxidative Stress-Induced Muscle Atrophy via Improvement of Mitochondrial Biogenesis

doi: 10.3390/antiox12081576

Figure Lengend Snippet: Effects of gRc on H 2 O 2 -induced degradation of myotubes. ( A ) C2C12 myotubes were treated with gRc for 24 h and cell viability was measured. ( B ) Myotubes were treated with H 2 O 2 up to 1 mM for 24 h. Relative cell viability and mitochondrial mass compared to vehicle-treated cells were expressed as the mean ± SEM ( n = 3). Dots are individual values. ( C ) Myotubes were pretreated with gRc for 12 h and further incubated with H 2 O 2 . After 24 h, cell viability and mitochondrial mass were determined and relative values were expressed as the mean ± SEM ( n = 3). Dots are individual values. ( D ) Myotubes were pretreated with gRc for 12 h and then treated with H 2 O 2 . After 24 h, cells were subjected to immunofluorescence staining for MyHC (green) and DAPI (blue). Fusion index and myotube length were quantitated and presented as the mean ± SEM ( n = 7). Dots are individual values. ( E ) Effects of gRc on the expression of MyHC, MyoD, MAFbx, and MuRF1 in H 2 O 2 -treated myotubes were determined by Western blotting. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. vehicle-treated cells, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. H 2 O 2 + vehicle-treated cells; n.s., non-significant. Scale bar = 100 μm.

Article Snippet: Murine skeletal muscle cell C2C12 myoblasts (CRL-1772; ATCC, Manassas, VA, USA) were cultured in a growth medium (GM, Dulbecco’s Modified Eagle Medium (DMEM) with 4.5 g/L glucose containing 10% heat-inactivated fetal bovine serum (FBS) and 100 IU penicillin/100 μg/mL streptomycin (P/S).

Techniques: Incubation, Immunofluorescence, Staining, Expressing, Western Blot

Journal: Antioxidants

Article Title: Ginsenoside Rc, an Active Component of Panax ginseng , Alleviates Oxidative Stress-Induced Muscle Atrophy via Improvement of Mitochondrial Biogenesis

doi: 10.3390/antiox12081576

Figure Lengend Snippet: Transcriptome analysis of the effect of gRc in H 2 O 2 -treated C2C12 myotubes. ( A ) Normalized enrichment score (NES) of the gene set enrichment analysis (GSEA) results of H 2 O 2 treatment compared to vehicle and the H 2 O 2 + gRc treatment compared to the H 2 O 2 treatment. The pathway terms were sourced from Gene Ontology—Biological Process (“Regulation of Oxidative Stress Induced Cell Death”, “Positive Regulation of Striated Muscle Cell Differentiation”, “ATP Synthesis Coupled Electron Transport”, “Muscle Cell Proliferation, Cellular Respiration”, and “Positive Regulation of Muscle Hypertrophy”), Gene Ontology—Cellular Component (“Mitochondrial Protein Containing Complex” and “Inner Mitochondrial Membrane Protein Complex”), Reactome (“Apoptosis” and “Myogenesis”), and WikiPathways (“Electron Transport Chain Oxphos System in Mitochondria”). * p < 0.05, ** p < 0.01. ( B ) The selected GSEA plots of H 2 O 2 treatment compared to vehicle (top) and the H 2 O 2 + gRc treatment compared to the H 2 O 2 treatment (bottom).

Article Snippet: Murine skeletal muscle cell C2C12 myoblasts (CRL-1772; ATCC, Manassas, VA, USA) were cultured in a growth medium (GM, Dulbecco’s Modified Eagle Medium (DMEM) with 4.5 g/L glucose containing 10% heat-inactivated fetal bovine serum (FBS) and 100 IU penicillin/100 μg/mL streptomycin (P/S).

Techniques: Cell Differentiation

Journal: Journal of Cellular and Molecular Medicine

Article Title: MRTF‐A regulates myoblast commitment to differentiation by targeting PAX7 during muscle regeneration

doi: 10.1111/jcmm.16820

Figure Lengend Snippet: RNA‐seq results reveal MRTF‐A knockdown down‐regulates/up‐regulates the skeletal muscle regulation involving genes in C2C12 cells. (A) The mRNA and protein levels of MRTF‐A in C2C12 cells transfected with shRNA lentivirus targeting MRTF‐A as compared to the shRNA‐control group. (B) Volcano plot of differentially expressed genes between sh‐MRTF‐A and shRNA‐control in C2C12 cells as determined by RNA‐seq. (C) Statistics of GO Enrichment analysis to categorize the pathways that are significantly altered upon MRTF‐A knockdown. The striated muscle thin filament, skeletal muscle contraction, regulation of muscle contraction and muscle contraction signalling are highlighted. (D) The heat map analysis showing the differentially regulated skeletal muscle development genes, data were presented as log 2 (FPKM+1). (E) Validation of identified muscle cell self‐renewal–related genes through FPKM (Reads Per Kilobase of exon model per Million mapped reads, FPKM ≥1) in MRTF‐A knockdown or control muscle cells. (F) Validation of identified muscle cell differentiation related genes through FPKM. * p < 0.05, ** p < 0.01

Article Snippet: The C3H murine skeletal muscle cell line C2C12 myoblasts (American Type Culture Collection, CRL‐1772) were cultured in growth medium (GM), which was consisting of Dulbecco's modified Eagle's medium (DMEM) (cat. no. 10569‐010, Gibco), 10% FBS (cat. no. 10099–141, Gibco) and 1% penicillin‐streptomycin (cat. no. SV30010, Hyclone) at 37°C under a humidified atmosphere with 5% CO 2 .

Techniques: RNA Sequencing, Knockdown, Transfection, shRNA, Control, Biomarker Discovery, Cell Differentiation

Journal: Journal of Cellular and Molecular Medicine

Article Title: MRTF‐A regulates myoblast commitment to differentiation by targeting PAX7 during muscle regeneration

doi: 10.1111/jcmm.16820

Figure Lengend Snippet: Up‐regulation of MRTF‐A and PAX7 during muscle cell differentiation and muscle regeneration. (A) MRTF‐A and PAX7 presented the same expression patterns during satellite cells (SCs) differentiation. The qPCR assay was conducted using sorted SCs, SCs maintained in growth medium (GM), and SCs at days 1, 2 or 3 of differentiation in differentiation medium (DM). (B) The mRNA expression of the MRTF ‐ A and PAX7 genes in differentiating C2C12 cells. (C) Haematoxylin‐eosin stain was performed to assess the muscle repair after CTX injection for 1, 5, 7 and 10 days. NS represented the normal saline‐treated group. Scale bar, 50 μm. (D) The MRTF‐A mRNA expression in NS control and muscle tissue after CTX injection for 0, 1, 3, 5, 7, 10 and 14 days. (E) The PAX7 mRNA expression in NS control and muscle tissue after CTX injection for 0, 1, 3, 5, 7, 10 and 14 days. (F) The protein level of the MRTF‐A and PAX7 in CTX‐injected tissues. (G) The densitometric quantification analysis of three independent Western blot experiments. The β‐actin was used to serve as a loading control. ** p < 0.01

Article Snippet: The C3H murine skeletal muscle cell line C2C12 myoblasts (American Type Culture Collection, CRL‐1772) were cultured in growth medium (GM), which was consisting of Dulbecco's modified Eagle's medium (DMEM) (cat. no. 10569‐010, Gibco), 10% FBS (cat. no. 10099–141, Gibco) and 1% penicillin‐streptomycin (cat. no. SV30010, Hyclone) at 37°C under a humidified atmosphere with 5% CO 2 .

Techniques: Cell Differentiation, Expressing, Staining, Injection, Saline, Control, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: MRTF‐A regulates myoblast commitment to differentiation by targeting PAX7 during muscle regeneration

doi: 10.1111/jcmm.16820

Figure Lengend Snippet: MRTF‐A promotes the proliferation of myoblast. (A) C2C12 myoblasts were transfected with pCDH‐vector or pCDH‐MRTF‐A, and cell proliferation was assessed using 5′‐Ethynyl‐2′‐deoxyuridine (EdU) assay. The scale bar represents 100 μm. (B) The percentage of EdU‐positive cells was analysed in figure (A). (C) The EdU assay was used to detect the cell proliferation of C2C12 cells that was transfected with shRNA‐control or shRNA‐MRTF‐A. The scale bar represents 100 μm. (D) The percentage of EdU‐positive cells was analysed in figure (C). Cell proliferation was detected using the cell counting kit‐8 (CCK‐8) assay in C2C12 cells with MRTF‐A overexpression (E) and MRTF‐A knockdown (F) comparing to control groups. (G, H) The mRNA levels of the proliferation marker gene CyclinD1 and PCNA were quantified using qPCR. Data are presented as means ± SEM for three independent experiments. * p < 0.05, ** p < 0.01.

Article Snippet: The C3H murine skeletal muscle cell line C2C12 myoblasts (American Type Culture Collection, CRL‐1772) were cultured in growth medium (GM), which was consisting of Dulbecco's modified Eagle's medium (DMEM) (cat. no. 10569‐010, Gibco), 10% FBS (cat. no. 10099–141, Gibco) and 1% penicillin‐streptomycin (cat. no. SV30010, Hyclone) at 37°C under a humidified atmosphere with 5% CO 2 .

Techniques: Transfection, Plasmid Preparation, EdU Assay, shRNA, Control, Cell Counting, CCK-8 Assay, Over Expression, Knockdown, Marker

Journal: Journal of Cellular and Molecular Medicine

Article Title: MRTF‐A regulates myoblast commitment to differentiation by targeting PAX7 during muscle regeneration

doi: 10.1111/jcmm.16820

Figure Lengend Snippet: MRTF‐A overexpression inhibits myoblast commitment towards differentiated cells. (A) Coimmunostaining for PAX7 (red) and MyoD (green) of C2C12 control cells, MRTF‐A knockdown and MRTF‐A overexpression C2C12 cells at 0, 12 and 24 h. The representative images of each group are shown. The inset photograph (large box) represented the higher magnification (200×) of the C2C12 cells from the small box. The scale bar represents 100 μm. (B) Percentage of PAX7 + /MyoD − , PAX7 + /MyoD + and PAX7 − /MyoD + cells were analysed during onset of the differentiation process in 15 different microscopic fields. The data show mean values of the percentage of cells on three different slides, error bars represent standard error of the mean. The experiment was repeated twice with similar results. (C) The mRNA expression of potential PAX7‐target genes in C2C12 cells. C2C12 cells were divided into three groups: stably transfected pCDH‐vector, stably transfected pCDH‐MRTF‐A, transfected siRNA‐PAX7 in stably transfected pCDH‐MRTF‐A cells. (D) The mRNA expression of potential PAX7‐target genes by rescuing of the MRTF‐A knockdown C2C12 cell with PAX7 overexpression (PAX7 OE). C2C12 cells were divided into three groups: stably transfected shRNA vector, stably transfected shRNA‐MRTF‐A, transfected pCDH‐PAX7 in stably transfected shRNA‐MRTF‐A cells. * p < 0.05, ** p < 0.01

Article Snippet: The C3H murine skeletal muscle cell line C2C12 myoblasts (American Type Culture Collection, CRL‐1772) were cultured in growth medium (GM), which was consisting of Dulbecco's modified Eagle's medium (DMEM) (cat. no. 10569‐010, Gibco), 10% FBS (cat. no. 10099–141, Gibco) and 1% penicillin‐streptomycin (cat. no. SV30010, Hyclone) at 37°C under a humidified atmosphere with 5% CO 2 .

Techniques: Over Expression, Control, Knockdown, Expressing, Stable Transfection, Transfection, Plasmid Preparation, shRNA

Journal: Journal of Cellular and Molecular Medicine

Article Title: MRTF‐A regulates myoblast commitment to differentiation by targeting PAX7 during muscle regeneration

doi: 10.1111/jcmm.16820

Figure Lengend Snippet: MRTF‐A inhibits the differentiation of myoblast. (A) The mRNA expression of MRTF‐A, PAX7, MyoD and MyoG in C2C12 cells transfected with pCDH‐vector or pCDH‐MRTF‐A. (B) Western blot for MRTF‐A, PAX7, MyoD and MyoG in C2C12 cells transfected with pCDH‐vector or pCDH‐MRTF‐A. (C, D) The mRNA and protein level of MRTF‐A, PAX7, MyoD and MyoG in C2C12 cells transfected with shRNA‐control and shRNA‐MRTF‐A. The densitometric quantification was analysed from three independent Western blot experiments (B, D). GAPDH expression was analysed to ensure equal loading of samples. (E) The differentiation of C2C12 cells stably overexpressing MRTF‐A was examined by staining for MyoG after 0 d (D0) and 3 d (D3) of culture in DM. Cells with an empty vector as the control. (F) The graph shows MyoG‐positive cells as a proportion of total cell number (shown by DAPI staining) for MRTF‐A‐overexpressing cells compared to control cells. (G) MyHC immunocytochemistry (red) for MRTF‐A overexpressing cells and control cells (pCDH‐vector) at D0 and D3 of differentiation. (H) Number of fibres per field of view and number of nuclei per fibre in images used for counting in (G), shown relative to control cells, * p < 0.05, ** p < 0.01

Article Snippet: The C3H murine skeletal muscle cell line C2C12 myoblasts (American Type Culture Collection, CRL‐1772) were cultured in growth medium (GM), which was consisting of Dulbecco's modified Eagle's medium (DMEM) (cat. no. 10569‐010, Gibco), 10% FBS (cat. no. 10099–141, Gibco) and 1% penicillin‐streptomycin (cat. no. SV30010, Hyclone) at 37°C under a humidified atmosphere with 5% CO 2 .

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, shRNA, Control, Stable Transfection, Staining, Immunocytochemistry

Journal: Journal of Cellular and Molecular Medicine

Article Title: MRTF‐A regulates myoblast commitment to differentiation by targeting PAX7 during muscle regeneration

doi: 10.1111/jcmm.16820

Figure Lengend Snippet: MRTF‐A regulates PAX7 expression by directly binding to the CArG box region of the PAX7 promoter. (A) Promoter activity of the PAX7 gene with WT, Cut‐1 (CArG box 1 cut‐down), Cut‐2 (CArG box 2 cut‐down), Mutation‐1 (CArG box 1 mutation) and Mutation‐2 (CArG box 2 mutation) promoters by dual‐luciferase reporter assay. (B) The effects of MRTF‐A on promoter activity of the PAX7 gene. The pCDH‐vector, pCDH‐MRTF‐A, shRNA‐control and shRNA‐MRTF‐A were co‐transfected with dual‐luciferase reporter plasmids, respectively. (C) The effects of MRTF‐A on promoter activity of the PAX7 gene with Cut‐1 or Cut‐2 promoter. The pCDH‐vector, pCDH‐MRTF‐A, shRNA‐control and shRNA‐MRTF‐A were co‐transfected with Cut‐1 or Cut‐2 plasmid, respectively. (D) Detection of interaction of MRTF‐A and CArG box 1 or CArG box 2 within the PAX7 promoter by EMSA. A complete set of three reactions was performed using the nuclear extracts prepared from normal C2C12 cells. The 200‐fold of unlabeled probes were used as specific competitors to demonstrate that the signal shift observed results from specific protein: DNA interaction. Arrowhead shows the specific complex. (E) The binding of the MRTF‐A on CArG box of the PAX7 gene promoter by ChIP assay. Histone H3 Antibody treatment was used as the positive control. (F) The percentage of input in IgG and MRTF‐A antibody–treated groups. * p < 0.05, ** p < 0.01

Article Snippet: The C3H murine skeletal muscle cell line C2C12 myoblasts (American Type Culture Collection, CRL‐1772) were cultured in growth medium (GM), which was consisting of Dulbecco's modified Eagle's medium (DMEM) (cat. no. 10569‐010, Gibco), 10% FBS (cat. no. 10099–141, Gibco) and 1% penicillin‐streptomycin (cat. no. SV30010, Hyclone) at 37°C under a humidified atmosphere with 5% CO 2 .

Techniques: Expressing, Binding Assay, Activity Assay, Mutagenesis, Luciferase, Reporter Assay, Plasmid Preparation, shRNA, Control, Transfection, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Low-Dose Acrolein, an Endogenous and Exogenous Toxic Molecule, Inhibits Glucose Transport via an Inhibition of Akt-Regulated GLUT4 Signaling in Skeletal Muscle Cells

doi: 10.3390/ijms22137228

Figure Lengend Snippet: Effects of acrolein on glucose uptake and GLUT4 protein expression in differentiated C2C12 myotubes. ( A ) Myotubes were treated with 1 μM acrolein in the presence or absence of insulin (10 nM) for 24 h and 72 h. The uptake of 2-NBDG into the myotubes was evaluated by a microplate fluorometer. ( B ) The GLUT4 protein expressions in myotubes treated with various concentrations of acrolein (0.5–2 μM) for 24 h are shown. ( C ) The GLUT4 protein expressions in myotubes treated with acrolein (1 μM) for 24 h and 72 h are shown. The protein expression was determined by Western blotting and quantified using densitometric analysis. Results are represented as means ± SEM for at least four independent experiments. * p < 0.05 versus vehicle control; # p < 0.05 versus acrolein alone.

Article Snippet: The murine skeletal muscle myoblast cell line C2C12 was purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Low-Dose Acrolein, an Endogenous and Exogenous Toxic Molecule, Inhibits Glucose Transport via an Inhibition of Akt-Regulated GLUT4 Signaling in Skeletal Muscle Cells

doi: 10.3390/ijms22137228

Figure Lengend Snippet: Acrolein interferes with glucose metabolic signaling molecules in differentiated C2C12 myotubes. Myotubes were treated with 1 μM acrolein for 24 h. ( A ) The levels of phosphorylated and total protein expression of IRS1, Akt, mTOR, p70S6K, and GSK3α/β were determined by Western blotting and quantified using densitometric analysis. ( B ) The levels of phosphorylated and total protein expression of p85/PI3K were determined by Western blotting and quantified using densitometric analysis. ( C ) The glycogen contents in myotubes treated with acrolein (1 μM) for 72 h are shown. Results are represented as means ± SEM for at least four independent experiments. * p < 0.05 versus vehicle control.

Article Snippet: The murine skeletal muscle myoblast cell line C2C12 was purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Low-Dose Acrolein, an Endogenous and Exogenous Toxic Molecule, Inhibits Glucose Transport via an Inhibition of Akt-Regulated GLUT4 Signaling in Skeletal Muscle Cells

doi: 10.3390/ijms22137228

Figure Lengend Snippet: Acrolein interferes with insulin signaling in differentiated C2C12 myotubes. Myotubes were treated with 1 μM acrolein for 24 h and then stimulated with insulin (10 nM) for 10 min. The levels of phosphorylated and total protein expression of Akt, GSK3α/β, IRS1, and IR were determined by Western blotting and quantified using densitometric analysis. Results are represented as means ± SEM for at least four independent experiments. * p < 0.05 versus insulin alone.

Article Snippet: The murine skeletal muscle myoblast cell line C2C12 was purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Western Blot