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ATCC murine skeletal muscle myoblast cell line
Murine Skeletal Muscle Myoblast Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 8620 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/murine+muscle+myoblast+cell+line+c2c12/C2C12/10__1097_slash_shk__0000000000002613-87-3-26
Average 99 stars, based on 8620 article reviews

murine skeletal muscle myoblast cell line - by Bioz Stars, 2026-09

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Article Title: PARP1 mediated PARylation contributes to myogenic progression and glucocorticoid transcriptional response.
Article Snippet: .. The murine muscle myoblast cell line C2C12 was purchased (ATCC, VA, USA) and 87 maintained in proliferation media, composed of Dulbecco’s Modified Eagle’s Medium 88 (DMEM) 25 mM glucose (Lonza, UK) supplemented with 10% (v/v) fetal bovine serum (FBS) 89 (Thermo, UK) and 1% Penicillin/Streptomycin (P/S) (Thermo, UK). .. Upon cells reaching 70 – 90 80% confluence, the differentiation medium, composed of DMEM 25 mM glucose 91 supplemented with 2% horse serum (HS) (Thermo, UK) and 1% P/S, was added to induce 92 differentiation.

Article Title: PARP1 mediated PARylation contributes to myogenic progression and glucocorticoid transcriptional response
Article Snippet: .. The murine muscle myoblast cell line C2C12 was purchased (ATCC, VA, USA) and maintained in proliferation media, composed of Dulbecco’s Modified Eagle’s Medium (DMEM) 25 mM glucose (Lonza, UK) supplemented with 10% (v/v) fetal bovine serum (FBS) (Thermo, UK) and 1% Penicillin/Streptomycin (P/S) (Thermo, UK). .. Upon cells reaching 70–80% confluence, the differentiation medium, composed of DMEM 25 mM glucose supplemented with 2% horse serum (HS) (Thermo, UK) and 1% P/S, was added to induce differentiation.

Image Search Results

A Western immunoblotting of differentiating C2C12 myoblasts probed for PARylation (PAR), PARP1, Myogenin, and Alpha-Tubulin (representative of n = 4). B Quantification of MYOG, PAR, and PARP1 present over differentiation. Each bar represents means ± S.D ( n = 4) *** P < 0.001. C Western immunoblotting of differentiating LHCN-M2 human myoblasts probed for MYOG, PAR, PARP1, Myogenin, and Alpha-Tubulin (representative of n = 4). D Quantification of PAR and PARP1 present over differentiation. Each bar represents means ± S.D ( n = 4) *** P < 0.001.

Journal: Cell Death Discovery

Article Title: PARP1 mediated PARylation contributes to myogenic progression and glucocorticoid transcriptional response

doi: 10.1038/s41420-023-01420-2

Figure Lengend Snippet: A Western immunoblotting of differentiating C2C12 myoblasts probed for PARylation (PAR), PARP1, Myogenin, and Alpha-Tubulin (representative of n = 4). B Quantification of MYOG, PAR, and PARP1 present over differentiation. Each bar represents means ± S.D ( n = 4) *** P < 0.001. C Western immunoblotting of differentiating LHCN-M2 human myoblasts probed for MYOG, PAR, PARP1, Myogenin, and Alpha-Tubulin (representative of n = 4). D Quantification of PAR and PARP1 present over differentiation. Each bar represents means ± S.D ( n = 4) *** P < 0.001.

Article Snippet: The murine muscle myoblast cell line C2C12 was purchased (ATCC, VA, USA) and maintained in proliferation media, composed of Dulbecco’s Modified Eagle’s Medium (DMEM) 25 mM glucose (Lonza, UK) supplemented with 10% (v/v) fetal bovine serum (FBS) (Thermo, UK) and 1% Penicillin/Streptomycin (P/S) (Thermo, UK).

Techniques: Western Blot

A , B Western immunoblotting of differentiating myoblasts differentiated in ±nicotinamide riboside (NR) (0.5 mM) (representative of n = 4) probed for PAR, PARP1, MYOD, and Alpha-Tubulin. C , D Western immunoblotting of differentiating myoblasts differentiated in ± NAMPT specific inhibitor FK866 (50 nM) (representative of n = 4) probed for PAR, PARP1, Myogenin, and Alpha-Tubulin, *** P < 0.001. E , F Western immunoblotting of differentiating myoblasts differentiated in ±dexamethasone (1 µM) probed for PAR, PARP1, MYOD and Alpha-Tubulin (representative of n = 4, ** P < 0.01). G , H C2C12 myoblasts differentiated in differentiation medium containing different glucose concentrations before lysate harvest and immunoblotted for PAR, PARP1, Hexokinase II, and Alpha-Tubulin (representative of n = 3, *P < 0.05). I , J Western immunoblotting of differentiating myoblasts differentiated in ±dexamethasone (1 µM), PARP inhibitor PJ34 (10 µM) or both in combination (representative of n = 3, * P < 0.05, ** P < 0.01).

Journal: Cell Death Discovery

Article Title: PARP1 mediated PARylation contributes to myogenic progression and glucocorticoid transcriptional response

doi: 10.1038/s41420-023-01420-2

Figure Lengend Snippet: A , B Western immunoblotting of differentiating myoblasts differentiated in ±nicotinamide riboside (NR) (0.5 mM) (representative of n = 4) probed for PAR, PARP1, MYOD, and Alpha-Tubulin. C , D Western immunoblotting of differentiating myoblasts differentiated in ± NAMPT specific inhibitor FK866 (50 nM) (representative of n = 4) probed for PAR, PARP1, Myogenin, and Alpha-Tubulin, *** P < 0.001. E , F Western immunoblotting of differentiating myoblasts differentiated in ±dexamethasone (1 µM) probed for PAR, PARP1, MYOD and Alpha-Tubulin (representative of n = 4, ** P < 0.01). G , H C2C12 myoblasts differentiated in differentiation medium containing different glucose concentrations before lysate harvest and immunoblotted for PAR, PARP1, Hexokinase II, and Alpha-Tubulin (representative of n = 3, *P < 0.05). I , J Western immunoblotting of differentiating myoblasts differentiated in ±dexamethasone (1 µM), PARP inhibitor PJ34 (10 µM) or both in combination (representative of n = 3, * P < 0.05, ** P < 0.01).

Techniques: Western Blot

A Western immunoblotting of differentiating C2C12 myoblasts differentiated in ±PARP inhibitor PJ34 (10 µM) probed for PAR, PARP1, Troponin 1 (TNNT1), and Alpha-Tubulin (representative of n = 4). B Quantification of PAR and PARP1 during differentiation. Each bar represents means ± S.D (representative of n = 4). C Western immunoblotting of differentiating LHCN-M2 myoblasts differentiated in ±PARP inhibitor PJ34 (10 µM) probed for PAR, PARP1, TNNT1, and Alpha-Tubulin ( n = 4) * P < 0.05, ** P < 0.01, and *** P < 0.001. D Quantification of PAR and PARP1 in human myoblasts during differentiation. Each bar represents means ± S.D (representative of n = 4). E Immunostaining of PAR (red), DAPI (purple), and MYOD (green) in differentiating C2C12 myoblasts ( n = 4). F Dot plot of gene ontology (GO) overrepresentation analysis of C2C12 myoblasts differentiated in ±PARP inhibitor PJ34 (10 µM) ( n = 3) on day 1 of differentiation. The x axis shows the gene ratio which represents the percentage of genes enriched in a term. The y axis represents the enriched pathways: size of the node represents the number of enriched genes in the term.

Journal: Cell Death Discovery

Article Title: PARP1 mediated PARylation contributes to myogenic progression and glucocorticoid transcriptional response

doi: 10.1038/s41420-023-01420-2

Figure Lengend Snippet: A Western immunoblotting of differentiating C2C12 myoblasts differentiated in ±PARP inhibitor PJ34 (10 µM) probed for PAR, PARP1, Troponin 1 (TNNT1), and Alpha-Tubulin (representative of n = 4). B Quantification of PAR and PARP1 during differentiation. Each bar represents means ± S.D (representative of n = 4). C Western immunoblotting of differentiating LHCN-M2 myoblasts differentiated in ±PARP inhibitor PJ34 (10 µM) probed for PAR, PARP1, TNNT1, and Alpha-Tubulin ( n = 4) * P < 0.05, ** P < 0.01, and *** P < 0.001. D Quantification of PAR and PARP1 in human myoblasts during differentiation. Each bar represents means ± S.D (representative of n = 4). E Immunostaining of PAR (red), DAPI (purple), and MYOD (green) in differentiating C2C12 myoblasts ( n = 4). F Dot plot of gene ontology (GO) overrepresentation analysis of C2C12 myoblasts differentiated in ±PARP inhibitor PJ34 (10 µM) ( n = 3) on day 1 of differentiation. The x axis shows the gene ratio which represents the percentage of genes enriched in a term. The y axis represents the enriched pathways: size of the node represents the number of enriched genes in the term.

Techniques: Western Blot, Immunostaining

A Heatmap showing 2911 detected proteins in differentiating C2C12 myoblasts differentiated in ± PARP inhibitor PJ34 (10 µM) over days 0, 1, 2, 3, 4, 5, and 6 of differentiation ( n = 3 per condition). B Volcano plots showing differentially abundant proteins in presence of PJ34 on each day of the 6 days of differentiation. Downregulated proteins are green (>−1.5 fold change), upregulated are red (>1.5 fold change). Arrows denote named proteins.

Journal: Cell Death Discovery

Article Title: PARP1 mediated PARylation contributes to myogenic progression and glucocorticoid transcriptional response

doi: 10.1038/s41420-023-01420-2

Figure Lengend Snippet: A Heatmap showing 2911 detected proteins in differentiating C2C12 myoblasts differentiated in ± PARP inhibitor PJ34 (10 µM) over days 0, 1, 2, 3, 4, 5, and 6 of differentiation ( n = 3 per condition). B Volcano plots showing differentially abundant proteins in presence of PJ34 on each day of the 6 days of differentiation. Downregulated proteins are green (>−1.5 fold change), upregulated are red (>1.5 fold change). Arrows denote named proteins.

Techniques:

A Schematic representation of the PARP inhibitor treatment protocol during C2C12 myoblast differentiation. B Heatmap representing differential abundance of proteins within samples differentiated in ±PARP1 specific inhibitor BYK204165 (10 µM) (Vehicle n = 7, BYK204165 n = 6). C Volcano plot of SWATH-LCMS lysates recovered from day 6 differentiated C2C12 myoblasts differentiated in ± PARP inhibitor PJ34 (10 µM). Differential protein abundance shown with downregulated proteins marked green and upregulated proteins marked red. D Myotube fusion index of differentiating myoblasts differentiated in ±PARP inhibitor PJ34 (10 µM) or ±PARP1 specific inhibitor BYK204165 (10 µM) ( n = 3). Cells were fixed on days 1, 3, and 6 of differentiation. Upper panel shows days 1, 3, and 6 fusion index, lower panel shows day 6 fusion index. Each bar represents means ± S.D ( n = 3) *** P < 0.001, ** P < 0.01. E Representative photographs of Jenner-Giemsa stained differentiating myoblasts over days 1, 3, and 6 of differentiation ( n = 3).

Journal: Cell Death Discovery

Article Title: PARP1 mediated PARylation contributes to myogenic progression and glucocorticoid transcriptional response

doi: 10.1038/s41420-023-01420-2

Figure Lengend Snippet: A Schematic representation of the PARP inhibitor treatment protocol during C2C12 myoblast differentiation. B Heatmap representing differential abundance of proteins within samples differentiated in ±PARP1 specific inhibitor BYK204165 (10 µM) (Vehicle n = 7, BYK204165 n = 6). C Volcano plot of SWATH-LCMS lysates recovered from day 6 differentiated C2C12 myoblasts differentiated in ± PARP inhibitor PJ34 (10 µM). Differential protein abundance shown with downregulated proteins marked green and upregulated proteins marked red. D Myotube fusion index of differentiating myoblasts differentiated in ±PARP inhibitor PJ34 (10 µM) or ±PARP1 specific inhibitor BYK204165 (10 µM) ( n = 3). Cells were fixed on days 1, 3, and 6 of differentiation. Upper panel shows days 1, 3, and 6 fusion index, lower panel shows day 6 fusion index. Each bar represents means ± S.D ( n = 3) *** P < 0.001, ** P < 0.01. E Representative photographs of Jenner-Giemsa stained differentiating myoblasts over days 1, 3, and 6 of differentiation ( n = 3).

Techniques: Data-independent acquisition, Quantitative Proteomics, Staining

A qPCR of PARP1 in scrambled sequence controls ( n = 5) and siRNA PARP1 transfected ( siPARP1 ) C2C12 myoblasts ( n = 4) *** P < 0.001. B Western immunoblotting of protein lysates collected from scrambled and siPARP1 C2C12 myoblasts ( n = 5). C Quantification of scrambled and siPARP1 transfections by western blotting ( n = 5). D qPCR of Scrambled control and siPARP1 cDNA for PARP1, PARP2/PARP2, PARG, NAMPT, and SIRT1 transcripts. Scrambled ( n = 5) and siPARP1 ( n = 4). E Representative volcano plot of differential gene expression following RNAseq of scrambled and siPARP1 C2C12 myoblasts. F Gene set enrichment analysis (GSEA) of pathways over and under-represented in RNAseq data of siPARP1 C2C12 myoblasts. G Enrichment plots of GSEA in siPARP1 C2C12 myoblasts for hypoxia, H myogenesis and, I TNFα via NF-κB.

Journal: Cell Death Discovery

Article Title: PARP1 mediated PARylation contributes to myogenic progression and glucocorticoid transcriptional response

doi: 10.1038/s41420-023-01420-2

Figure Lengend Snippet: A qPCR of PARP1 in scrambled sequence controls ( n = 5) and siRNA PARP1 transfected ( siPARP1 ) C2C12 myoblasts ( n = 4) *** P < 0.001. B Western immunoblotting of protein lysates collected from scrambled and siPARP1 C2C12 myoblasts ( n = 5). C Quantification of scrambled and siPARP1 transfections by western blotting ( n = 5). D qPCR of Scrambled control and siPARP1 cDNA for PARP1, PARP2/PARP2, PARG, NAMPT, and SIRT1 transcripts. Scrambled ( n = 5) and siPARP1 ( n = 4). E Representative volcano plot of differential gene expression following RNAseq of scrambled and siPARP1 C2C12 myoblasts. F Gene set enrichment analysis (GSEA) of pathways over and under-represented in RNAseq data of siPARP1 C2C12 myoblasts. G Enrichment plots of GSEA in siPARP1 C2C12 myoblasts for hypoxia, H myogenesis and, I TNFα via NF-κB.

Techniques: Sequencing, Transfection, Western Blot, Control, Gene Expression

A Venn diagram of genes positively regulated by glucocorticoids in Scrambled and siPARP1 C2C12 myoblasts (>1.5 Fold Change, <0.05 FDR) ( n = 5 per treatment). B Selected list of genes responsive to dexamethasone and gene expression lost in siPARP1 C2C12 myoblasts. C Manhattan plot produced following g:GOSt analysis using the 86 genes whose response to dexamethasone is lost in siPARP1 C2C12 myoblasts. The plot shows overrepresented processes by Molecular function (red), Biological process (orange), Cellular compartment (green), Transcription factors (blue), and Human phenotype (purple).

Journal: Cell Death Discovery

Article Title: PARP1 mediated PARylation contributes to myogenic progression and glucocorticoid transcriptional response

doi: 10.1038/s41420-023-01420-2

Figure Lengend Snippet: A Venn diagram of genes positively regulated by glucocorticoids in Scrambled and siPARP1 C2C12 myoblasts (>1.5 Fold Change, <0.05 FDR) ( n = 5 per treatment). B Selected list of genes responsive to dexamethasone and gene expression lost in siPARP1 C2C12 myoblasts. C Manhattan plot produced following g:GOSt analysis using the 86 genes whose response to dexamethasone is lost in siPARP1 C2C12 myoblasts. The plot shows overrepresented processes by Molecular function (red), Biological process (orange), Cellular compartment (green), Transcription factors (blue), and Human phenotype (purple).

Techniques: Gene Expression, Produced

Dose–response curve for the determination of the IC 50 value of JB006 for the inhibition of the cytotoxic activity of myotoxin II in C2C12 cells. Myotoxin II (10 μg; 0.73 nmol; 7.3 μM) was pre-incubated for 30 min at 37 °C with 900, 225, 56.25, 14.06, 3.52, or 0 μM JB006 in assay medium and added in triplicate wells. After an incubation of 3 h at 37 °C, the activity of LDH released by damaged cells was measured on the supernatant. 100% release corresponds to cells incubated with the toxin in the absence of the inhibitory peptide. Results are presented as mean ± SD ( n = 3).

Journal: ACS Omega

Article Title: In Vivo Neutralization of Myotoxin II, a Phospholipase A 2 Homologue from Bothrops asper Venom, Using Peptides Discovered via Phage Display Technology

doi: 10.1021/acsomega.2c00280

Figure Lengend Snippet: Dose–response curve for the determination of the IC 50 value of JB006 for the inhibition of the cytotoxic activity of myotoxin II in C2C12 cells. Myotoxin II (10 μg; 0.73 nmol; 7.3 μM) was pre-incubated for 30 min at 37 °C with 900, 225, 56.25, 14.06, 3.52, or 0 μM JB006 in assay medium and added in triplicate wells. After an incubation of 3 h at 37 °C, the activity of LDH released by damaged cells was measured on the supernatant. 100% release corresponds to cells incubated with the toxin in the absence of the inhibitory peptide. Results are presented as mean ± SD ( n = 3).

Article Snippet: The ATCC-CRL1772 murine myogenic cell line C2C12 was used to evaluate neutralization of the cytotoxic action of myotoxin II, as previously described.

Techniques: Inhibition, Activity Assay, Incubation

Inhibition of the cytotoxic activity of myotoxin II by different peptides (JB001–JB006). C2C12 myotubes were used. Myotoxin II (10 μg; 0.73 nmol; 7.3 μM) was pre-incubated for 30 min at 37 °C with 900 μM of each peptide in assay medium or without peptides and added in triplicate wells. After an incubation of 3 h at 37 °C, the activity of LDH released by damaged cells was measured on the supernatant. 100% release corresponds to cells incubated with 0.1% Triton X-100. Results are presented as mean ± SD ( n = 3). Incubation of myotoxin II with JB006 caused a significant reduction of LDH release (4 ± 5%) compared to cells treated with myotoxin II alone (87 ± 11%). P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****).

Journal: ACS Omega

Article Title: In Vivo Neutralization of Myotoxin II, a Phospholipase A 2 Homologue from Bothrops asper Venom, Using Peptides Discovered via Phage Display Technology

doi: 10.1021/acsomega.2c00280

Figure Lengend Snippet: Inhibition of the cytotoxic activity of myotoxin II by different peptides (JB001–JB006). C2C12 myotubes were used. Myotoxin II (10 μg; 0.73 nmol; 7.3 μM) was pre-incubated for 30 min at 37 °C with 900 μM of each peptide in assay medium or without peptides and added in triplicate wells. After an incubation of 3 h at 37 °C, the activity of LDH released by damaged cells was measured on the supernatant. 100% release corresponds to cells incubated with 0.1% Triton X-100. Results are presented as mean ± SD ( n = 3). Incubation of myotoxin II with JB006 caused a significant reduction of LDH release (4 ± 5%) compared to cells treated with myotoxin II alone (87 ± 11%). P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****).

Article Snippet: The ATCC-CRL1772 murine myogenic cell line C2C12 was used to evaluate neutralization of the cytotoxic action of myotoxin II, as previously described.

Techniques: Inhibition, Activity Assay, Incubation

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

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

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

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

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

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.

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.

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

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

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

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

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

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

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

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

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

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

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.

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

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.

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

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.

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

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