Journal: Molecular Therapy. Nucleic Acids

Article Title: Restoration of myogenesis in ALS-myocytes through miR-26a-5p-mediated Smad4 inhibition and its impact on motor neuron development

doi: 10.1016/j.omtn.2025.102581

Figure Lengend Snippet: Luciferase reporter assay and miRNA activity in C2C12 cells Luciferase reporter assays were performed to demonstrate the direct interaction between miRNAs and their targets. Part of the 3′-UTR sequence containing the miRNA putative interaction site (or not containing; Ctrl) was cloned in pmirGLO vector. Firefly luciferase (reporter gene) and Renilla luciferase (control reporter for normalization) activities were measured after the transfection in C2C12 cells together with miRNA mimics or miRNA scramble sequence (scramble). (A–C) Targets of miR-134-5p (A), miR-26a-5p (B), and miR-882 (C) showed a decreased luciferase activity. (D) After cell culture for 24 h with the medium containing miRNA mimics and 24 h of recovery, miRNAs within the cells increased their expression more than two times in comparison with the cells cultured with scramble sequence. Expression of miRNA targets validated by luciferase reporter assays decreased when the expression of miRNAs increased. (E–G) The expression of Creb1 decreased about twice (E), MyoD1 and Pax7 by about 50 percent (F), and Igfbp5 showed a decrease in its expression by one-third (G). Data are expressed as the mean of at least four independent transfections. Error bars indicate SD. p values from t tests considering independent variance between two groups in analysis are indicated as follows: ∗ p < 0.05, ∗∗ p < 0.001, and ∗∗∗ p < 0.0001.

Article Snippet: C2C12 myoblasts were cultured in Dulbecco’s modified Eagle’s medium with high glucose (Life Technologies) + 10% FBS (Life Technologies) in 10-cm dishes and split every 2 or 3 days before they reached 70% of confluence.

Techniques: Luciferase, Reporter Assay, Activity Assay, Sequencing, Clone Assay, Plasmid Preparation, Control, Transfection, Cell Culture, Expressing, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: Restoration of myogenesis in ALS-myocytes through miR-26a-5p-mediated Smad4 inhibition and its impact on motor neuron development

doi: 10.1016/j.omtn.2025.102581

Figure Lengend Snippet: Activity of miRNAs on motor neuron cells (A) Representative immunohistochemical images of motor neuron cultured with hSOD1(WT) (MCM WT) or hSOD1(G93A) PCM (MCM G93A). In blue, it is indicated cell nuclei and in red, motor neuron body and dendrites. (B) Calculation of neurite length. It decreases when hSOD1(G93A) PCM-derived culture medium was added to motor neurons. (C) Representative immunohistochemical images of motor neuron cells transfected with an empty pCMVmiR-GFP vector (empty) or a pCMVmiR-26a-GFP vector (miR-26a-5p) are presented. Green indicates transfected cells (GFP positive), blue indicates nuclei, and red represents the motor neuron body and neurites. (D) Only transfected cells (GFP-positive) were analyzed for neurite length, demonstrating an increase in neurite length when miR-26a is overexpressed. (E) Representative immunohistochemical images of motor neurons cultured in medium derived from C2C12 cells (MC C2C12) overexpressing miR-26a-5p, miR-134-5p, and miR-431-5p are presented. Red staining highlights the bodies and neurites of motor neurons, while blue staining depicts their nuclei. (F) Quantitative assessment of neurite length in motor neurons treated with culture medium derived from C2C12 cells overexpressing miR-26a-5p, miR-134-5p, and miR-431-5p. The miR-26a-5p and miR-431-5p miRNAs exhibit a stimulatory effect on neurite elongation. (G) Representative image of motor neuron neurite branching after miR-26a-5p overexpression. (H) Representative image of motor neuron neurite branching of motor neurons cultured in medium derived from C2C12 cells (MC C2C12) overexpressing miR-26a-5p. In both cases neurites appeared branched and several neurites outgrowth from cell body. (I) Quantification of neurites per body cell. The average number of neurites per 20 cells is represented. It increases when miR-26a is expressed from the cells (miR-26a modulation) or is present in the medium derived from C2C12 overexpressing miR-26a (MC C2C12). ∗∗ p < 0.005 and ∗∗∗ p < 0.0005, calculated according to t test between indicated samples considering unequal variance. SD is indicated considering at least three biological replicates and 15 cells analyzed per biological replicate.

Article Snippet: C2C12 myoblasts were cultured in Dulbecco’s modified Eagle’s medium with high glucose (Life Technologies) + 10% FBS (Life Technologies) in 10-cm dishes and split every 2 or 3 days before they reached 70% of confluence.

Techniques: Activity Assay, Immunohistochemical staining, Cell Culture, Derivative Assay, Transfection, Plasmid Preparation, Staining, Over Expression

Journal: International Journal of Molecular Medicine

Article Title: GW8510 alleviates muscle atrophy and skeletal muscle dysfunction in mice through AMPK/PGC1α signaling

doi: 10.3892/ijmm.2025.5569

Figure Lengend Snippet: GW8510 ameliorates DEX-induced atrophy in C2C12 myotubes in vitro . (A) Viability of C2C12 myoblasts treated with GW8510 for 24 h. n=5/group. (B) Relative expression of muscle atrophy-associated genes (Fbxo32 and Trim63) in C2C12 myotubes. n=3/group. (C) Western blot analysis of muscle atrophy-associated protein (Fbxo32, and Trim63). (D) Hematoxylin and eosin staining and quantification of diameter of C2C12 myotubes. Scale bar, 50 μ m. n=6/group. (E) Relative expression of muscle atrophy-associated genes (Myog, Fbxo32 and Trim63). (F) Expression levels of muscle atrophy-related protein (Myog, Fbxo32 Trim63). n=3/group. (G) SOD and creatine kinase activity. n=4/group. (H) Relative expression of muscle fibrosis-related genes (Acta2 and Tgfb) in C2C12 myotubes stimulated with DEX (10 μ M) or vehicle, followed by treatment with GW8510 (2 μ M) or vehicle for 24 h. n=3/group. # P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001 vs. control; * P<0.05, ** P<0.01, *** P<0.001 vs. DEX. DEX, dexamethasone; Fbxo, F-box protein; Trim, tripartite motif; SOD, superoxide dismutase.

Article Snippet: Mouse C2C12 myoblasts were purchased from Procell Life Science & Technology Co., Ltd. and authenticated by STR profiling.

Techniques: In Vitro, Expressing, Western Blot, Staining, Activity Assay, Control

Journal: International Journal of Molecular Medicine

Article Title: GW8510 alleviates muscle atrophy and skeletal muscle dysfunction in mice through AMPK/PGC1α signaling

doi: 10.3892/ijmm.2025.5569

Figure Lengend Snippet: GW8510 improves mitochondrial function and biogenesis in C2C12 in vitro . (A) Reactive oxygen species staining using DCFH-DA in C2C12 myotubes stimulated with DEX (10 μ M) or vehicle, followed by treatment with GW8510 (2 μ M) or vehicle for 24 h. Scale bar, 20 μ m. (B) Relative fluorescence intensity in C2C12 myotubes using ImageJ. n=5/group. (C) Relative mitochondrial mass using Mitotracker DeepRed staining. n=8/group. (D) Relative mtDNA copy number in C2C12 myotubes stimulated with DEX (10 μ M) or vehicle, followed by treatment with GW8510 (2 μ M) or vehicle for 24 h. n=4/group. (E) Western blot of mitochondrial fission and fusion-related protein (Opa1, Mfn1, Drp1 and p-Drp1). (F) Relative expression of mitochondrial biogenesis-related genes (Tfam, Sirt1, Nrf1 and Pgc1α) in C2C12 myotubes stimulated with DEX (10 μ M) or vehicle, followed by treatment with GW8510 (2 μ M) or vehicle for 24 h. n=3/group. # P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001 vs. control; * P<0.05, ** P<0.01, **** P<0.0001 vs. DEX. DEX, dexamethasone; mtDNA, mitochondrial DNA; Mfn, mitofusin; p-Drp, phosphorylated dynamin-related protein; Tfam, transcription factor A, mitochondrial; Sirt1, sirtuin 1; Pgc, peroxisome proliferator-activated receptor gamma, coactivator.

Article Snippet: Mouse C2C12 myoblasts were purchased from Procell Life Science & Technology Co., Ltd. and authenticated by STR profiling.

Techniques: In Vitro, Staining, Fluorescence, Western Blot, Expressing, Control

Journal: International Journal of Molecular Medicine

Article Title: GW8510 alleviates muscle atrophy and skeletal muscle dysfunction in mice through AMPK/PGC1α signaling

doi: 10.3892/ijmm.2025.5569

Figure Lengend Snippet: GW8510 increases NAD + levels and ATP production and protects against oxidative stress. (A) Levels of NAD + and NADH, and the ratio of NAD + /NADH in C2C12 myotubes. n=4/group. Concentration of (B) ATP and (C) MDA in C2C12 myotubes stimulated with DEX (10 μ M) or vehicle, followed by treatment with GW8510 (2 μ M) or vehicle for 24 h. (D) Levels of NAD + and NADH, and the ratio of NAD + /NADH in GC tissue. Concentration of (E) ATP and (F) MDA in GC tissue. (G). Activity of GSH in GC tissue. n=4/group. (H) Activity of GSH in serum. n=5/group. # P<0.05, ## P<0.01, ### P<0.001 vs. control/sham; * P<0.05, ** P<0.01, *** P<0.001 vs. DEX/vehicle. MDA, malondialdehyde; DEX, dexamethasone; GC, gastrocnemius; GSH, glutathione; prot, protein.

Article Snippet: Mouse C2C12 myoblasts were purchased from Procell Life Science & Technology Co., Ltd. and authenticated by STR profiling.

Techniques: Concentration Assay, Activity Assay, Control

Journal: International Journal of Molecular Medicine

Article Title: GW8510 alleviates muscle atrophy and skeletal muscle dysfunction in mice through AMPK/PGC1α signaling

doi: 10.3892/ijmm.2025.5569

Figure Lengend Snippet: GW8510 exerts a protective effect on expression of genes associated with muscle atrophy and development. (A) Principal component analysis score plot on C2C12 myotubes stimulated with DEX (10 μ M) or vehicle, followed by treatment with GW8510 (2 μ M) or vehicle for 24 h. (B) Overlap between genes down- and upregulated by dexamethasone and GW8510. (C) Heatmap of muscle atrophy and development-associated genes, including mitochondrial dynamics- (Fis1, Mfn1, Mfn2 and Opa1), antioxidation-related genes (Sod2, Sod1 and Cat), myoblast differentiation-related genes (Myf5, MyF6 and Myof), mitophagy-related genes (Sqstm1, Pink1, Map1lc3b, Atg12, Atg5 and Bnip4) and denervation-related genes (Chrna1, Gadd45a, Ncam1 and Musk). (D) Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis for significantly differentially expressed genes. (E) Validation of relative mRNA expression of muscle atrophy- and development-associated genes. n=3/group. ## P<0.01 vs. control; ** P<0.01 vs. DEX. DEX, dexamethasone; PC, principal component; Fis, fission, mitochondrial 1; Mfn, mitofusin; Opa1, OPA1 mitochondrial dynamin like GTPase; Sod, superoxide dismutase; Cat, catalase; Myf, myogenic factor; Myof, myoferlin; Gadd45a, growth arrest and DNA damage inducible alpha; Ncam, neural cell adhesion molecule; Chma, cholinergic receptor nicotinic α; Pink, PTEN induced kinase; Sqstm, sequestosome; Map1lc3b, microtubule associated protein 1 light chain 3 beta; Atg, autophagy related; Bnip, BCL2 interacting protein; Pgc, peroxisome proliferator-activated receptor γ, coactivator.

Article Snippet: Mouse C2C12 myoblasts were purchased from Procell Life Science & Technology Co., Ltd. and authenticated by STR profiling.

Techniques: Expressing, Biomarker Discovery, Control

Journal: International Journal of Molecular Medicine

Article Title: GW8510 alleviates muscle atrophy and skeletal muscle dysfunction in mice through AMPK/PGC1α signaling

doi: 10.3892/ijmm.2025.5569

Figure Lengend Snippet: GW8510 activates AMPK signaling cascade in C2C12 myotubes and sciatic nerve denervation mice. (A) Western blotting of Mstn, AMPKα and p-AMPKα and quantification of relative protein levels in C2C12 myotubes stimulated with DEX (10 μ M) or vehicle, followed by treatment with GW8510 (2 μ M) or vehicle for 24 h. n=3/group. (B) Relative expression of muscle atrophy-related genes (Myog, Fbxo32 and Trim63) in GC and SOL tissue. n=3-4/group. (C) Western blot of muscle atrophy- (Myog, Fbxo32, Trim63) and mitochondrial fission- and fusion-related protein (Opa1, Mfn1and p-Drp1) and Mstn, AMPK, and p-AMPK in GC tissue in non-denervated, denervated and denervated mice treated with GW8510. (D) Relative protein levels. n=3/group. # P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001 vs. control/sham; * P<0.05, ** P<0.01, *** P<0.001 vs. DEX/vehicle. Mstn, myostatin; p-, phosphorylated; DEX, dexamethasone; Myog, myogenin; Fbxo, F-box protein; Trim, tripartite motif; GC, gastrocnemius; SOL, soleus; Opa1, OPA1 mitochondrial dynamin like GTPase; Mfn, mitofusin; Drp, Dynamin related protein.

Article Snippet: Mouse C2C12 myoblasts were purchased from Procell Life Science & Technology Co., Ltd. and authenticated by STR profiling.

Techniques: Western Blot, Expressing, Control

Journal: International Journal of Molecular Medicine

Article Title: GW8510 alleviates muscle atrophy and skeletal muscle dysfunction in mice through AMPK/PGC1α signaling

doi: 10.3892/ijmm.2025.5569

Figure Lengend Snippet: GW8510 alleviates muscle atrophy via the AMPK/PGC1α signaling cascade. (A) mRNA and (B) protein expression of Cdk2 in C2C12 myotubes and in GC tissue. (C). Protein expression of Pgc1α in GC tissue. (D) Western blotting of Pgc1α in C2C12 myotubes after transfection of siRNAs targeting Pgc1α. Western blotting of Pgc1α, Fbxo32, Trim63, Mfn1, Drp1, p-Drp1, Myog and Mstn in C2C12 myotubes transfected with siRNA targeting Pgc1α, followed by stimulation with (E) DEX (10 μ M) or (F) TNFα (20 ng/ml) or vehicle and treatment with GW8510 (2 μ M) or vehicle for 24 h. n=3/group. # P<0.05, ## P<0.01, ### P<0.001 vs. control/sham; * P<0.05, *** P<0.001, **** P<0.0001 vs. DEX/vehicle. PGC, peroxisome proliferator-activated receptor γ, coactivator; Cdk, cyclin dependent kinase; GC, gastrocnemius; si, Small Interfering; Fbxo, F-box protein; Trim, tripartite motif; Mfn, mitofusin; p-Drp1, phosphorylated Dynamin related protein 1; Myog, myogenin; Mstn, myostatin; DEX, dexamethasone; NC, Negative control.

Article Snippet: Mouse C2C12 myoblasts were purchased from Procell Life Science & Technology Co., Ltd. and authenticated by STR profiling.

Techniques: Expressing, Western Blot, Transfection, Control, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: GW8510 alleviates muscle atrophy and skeletal muscle dysfunction in mice through AMPK/PGC1α signaling

doi: 10.3892/ijmm.2025.5569

Figure Lengend Snippet: GW8510 ameliorates DEX-induced atrophy in C2C12 myotubes in vitro . (A) Viability of C2C12 myoblasts treated with GW8510 for 24 h. n=5/group. (B) Relative expression of muscle atrophy-associated genes (Fbxo32 and Trim63) in C2C12 myotubes. n=3/group. (C) Western blot analysis of muscle atrophy-associated protein (Fbxo32, and Trim63). (D) Hematoxylin and eosin staining and quantification of diameter of C2C12 myotubes. Scale bar, 50 μ m. n=6/group. (E) Relative expression of muscle atrophy-associated genes (Myog, Fbxo32 and Trim63). (F) Expression levels of muscle atrophy-related protein (Myog, Fbxo32 Trim63). n=3/group. (G) SOD and creatine kinase activity. n=4/group. (H) Relative expression of muscle fibrosis-related genes (Acta2 and Tgfb) in C2C12 myotubes stimulated with DEX (10 μ M) or vehicle, followed by treatment with GW8510 (2 μ M) or vehicle for 24 h. n=3/group. # P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001 vs. control; * P<0.05, ** P<0.01, *** P<0.001 vs. DEX. DEX, dexamethasone; Fbxo, F-box protein; Trim, tripartite motif; SOD, superoxide dismutase.

Article Snippet: Viability of C2C12 myoblasts after GW8510 treatment was performed using CCK-8 (Shanghai Yeasen Biotechnology Co., Ltd.; cat. no. 40203ES60).

Techniques: In Vitro, Expressing, Western Blot, Staining, Activity Assay, Control

Journal: International Journal of Molecular Medicine

Article Title: GW8510 alleviates muscle atrophy and skeletal muscle dysfunction in mice through AMPK/PGC1α signaling

doi: 10.3892/ijmm.2025.5569

Figure Lengend Snippet: GW8510 improves mitochondrial function and biogenesis in C2C12 in vitro . (A) Reactive oxygen species staining using DCFH-DA in C2C12 myotubes stimulated with DEX (10 μ M) or vehicle, followed by treatment with GW8510 (2 μ M) or vehicle for 24 h. Scale bar, 20 μ m. (B) Relative fluorescence intensity in C2C12 myotubes using ImageJ. n=5/group. (C) Relative mitochondrial mass using Mitotracker DeepRed staining. n=8/group. (D) Relative mtDNA copy number in C2C12 myotubes stimulated with DEX (10 μ M) or vehicle, followed by treatment with GW8510 (2 μ M) or vehicle for 24 h. n=4/group. (E) Western blot of mitochondrial fission and fusion-related protein (Opa1, Mfn1, Drp1 and p-Drp1). (F) Relative expression of mitochondrial biogenesis-related genes (Tfam, Sirt1, Nrf1 and Pgc1α) in C2C12 myotubes stimulated with DEX (10 μ M) or vehicle, followed by treatment with GW8510 (2 μ M) or vehicle for 24 h. n=3/group. # P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001 vs. control; * P<0.05, ** P<0.01, **** P<0.0001 vs. DEX. DEX, dexamethasone; mtDNA, mitochondrial DNA; Mfn, mitofusin; p-Drp, phosphorylated dynamin-related protein; Tfam, transcription factor A, mitochondrial; Sirt1, sirtuin 1; Pgc, peroxisome proliferator-activated receptor gamma, coactivator.

Article Snippet: Viability of C2C12 myoblasts after GW8510 treatment was performed using CCK-8 (Shanghai Yeasen Biotechnology Co., Ltd.; cat. no. 40203ES60).

Techniques: In Vitro, Staining, Fluorescence, Western Blot, Expressing, Control

Journal: International Journal of Molecular Medicine

Article Title: GW8510 alleviates muscle atrophy and skeletal muscle dysfunction in mice through AMPK/PGC1α signaling

doi: 10.3892/ijmm.2025.5569

Figure Lengend Snippet: GW8510 increases NAD + levels and ATP production and protects against oxidative stress. (A) Levels of NAD + and NADH, and the ratio of NAD + /NADH in C2C12 myotubes. n=4/group. Concentration of (B) ATP and (C) MDA in C2C12 myotubes stimulated with DEX (10 μ M) or vehicle, followed by treatment with GW8510 (2 μ M) or vehicle for 24 h. (D) Levels of NAD + and NADH, and the ratio of NAD + /NADH in GC tissue. Concentration of (E) ATP and (F) MDA in GC tissue. (G). Activity of GSH in GC tissue. n=4/group. (H) Activity of GSH in serum. n=5/group. # P<0.05, ## P<0.01, ### P<0.001 vs. control/sham; * P<0.05, ** P<0.01, *** P<0.001 vs. DEX/vehicle. MDA, malondialdehyde; DEX, dexamethasone; GC, gastrocnemius; GSH, glutathione; prot, protein.

Article Snippet: Viability of C2C12 myoblasts after GW8510 treatment was performed using CCK-8 (Shanghai Yeasen Biotechnology Co., Ltd.; cat. no. 40203ES60).

Techniques: Concentration Assay, Activity Assay, Control

Journal: International Journal of Molecular Medicine

Article Title: GW8510 alleviates muscle atrophy and skeletal muscle dysfunction in mice through AMPK/PGC1α signaling

doi: 10.3892/ijmm.2025.5569

Figure Lengend Snippet: GW8510 exerts a protective effect on expression of genes associated with muscle atrophy and development. (A) Principal component analysis score plot on C2C12 myotubes stimulated with DEX (10 μ M) or vehicle, followed by treatment with GW8510 (2 μ M) or vehicle for 24 h. (B) Overlap between genes down- and upregulated by dexamethasone and GW8510. (C) Heatmap of muscle atrophy and development-associated genes, including mitochondrial dynamics- (Fis1, Mfn1, Mfn2 and Opa1), antioxidation-related genes (Sod2, Sod1 and Cat), myoblast differentiation-related genes (Myf5, MyF6 and Myof), mitophagy-related genes (Sqstm1, Pink1, Map1lc3b, Atg12, Atg5 and Bnip4) and denervation-related genes (Chrna1, Gadd45a, Ncam1 and Musk). (D) Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis for significantly differentially expressed genes. (E) Validation of relative mRNA expression of muscle atrophy- and development-associated genes. n=3/group. ## P<0.01 vs. control; ** P<0.01 vs. DEX. DEX, dexamethasone; PC, principal component; Fis, fission, mitochondrial 1; Mfn, mitofusin; Opa1, OPA1 mitochondrial dynamin like GTPase; Sod, superoxide dismutase; Cat, catalase; Myf, myogenic factor; Myof, myoferlin; Gadd45a, growth arrest and DNA damage inducible alpha; Ncam, neural cell adhesion molecule; Chma, cholinergic receptor nicotinic α; Pink, PTEN induced kinase; Sqstm, sequestosome; Map1lc3b, microtubule associated protein 1 light chain 3 beta; Atg, autophagy related; Bnip, BCL2 interacting protein; Pgc, peroxisome proliferator-activated receptor γ, coactivator.

Article Snippet: Viability of C2C12 myoblasts after GW8510 treatment was performed using CCK-8 (Shanghai Yeasen Biotechnology Co., Ltd.; cat. no. 40203ES60).

Techniques: Expressing, Biomarker Discovery, Control

Journal: International Journal of Molecular Medicine

Article Title: GW8510 alleviates muscle atrophy and skeletal muscle dysfunction in mice through AMPK/PGC1α signaling

doi: 10.3892/ijmm.2025.5569

Figure Lengend Snippet: GW8510 activates AMPK signaling cascade in C2C12 myotubes and sciatic nerve denervation mice. (A) Western blotting of Mstn, AMPKα and p-AMPKα and quantification of relative protein levels in C2C12 myotubes stimulated with DEX (10 μ M) or vehicle, followed by treatment with GW8510 (2 μ M) or vehicle for 24 h. n=3/group. (B) Relative expression of muscle atrophy-related genes (Myog, Fbxo32 and Trim63) in GC and SOL tissue. n=3-4/group. (C) Western blot of muscle atrophy- (Myog, Fbxo32, Trim63) and mitochondrial fission- and fusion-related protein (Opa1, Mfn1and p-Drp1) and Mstn, AMPK, and p-AMPK in GC tissue in non-denervated, denervated and denervated mice treated with GW8510. (D) Relative protein levels. n=3/group. # P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001 vs. control/sham; * P<0.05, ** P<0.01, *** P<0.001 vs. DEX/vehicle. Mstn, myostatin; p-, phosphorylated; DEX, dexamethasone; Myog, myogenin; Fbxo, F-box protein; Trim, tripartite motif; GC, gastrocnemius; SOL, soleus; Opa1, OPA1 mitochondrial dynamin like GTPase; Mfn, mitofusin; Drp, Dynamin related protein.

Article Snippet: Viability of C2C12 myoblasts after GW8510 treatment was performed using CCK-8 (Shanghai Yeasen Biotechnology Co., Ltd.; cat. no. 40203ES60).

Techniques: Western Blot, Expressing, Control

Journal: International Journal of Molecular Medicine

Article Title: GW8510 alleviates muscle atrophy and skeletal muscle dysfunction in mice through AMPK/PGC1α signaling

doi: 10.3892/ijmm.2025.5569

Figure Lengend Snippet: GW8510 alleviates muscle atrophy via the AMPK/PGC1α signaling cascade. (A) mRNA and (B) protein expression of Cdk2 in C2C12 myotubes and in GC tissue. (C). Protein expression of Pgc1α in GC tissue. (D) Western blotting of Pgc1α in C2C12 myotubes after transfection of siRNAs targeting Pgc1α. Western blotting of Pgc1α, Fbxo32, Trim63, Mfn1, Drp1, p-Drp1, Myog and Mstn in C2C12 myotubes transfected with siRNA targeting Pgc1α, followed by stimulation with (E) DEX (10 μ M) or (F) TNFα (20 ng/ml) or vehicle and treatment with GW8510 (2 μ M) or vehicle for 24 h. n=3/group. # P<0.05, ## P<0.01, ### P<0.001 vs. control/sham; * P<0.05, *** P<0.001, **** P<0.0001 vs. DEX/vehicle. PGC, peroxisome proliferator-activated receptor γ, coactivator; Cdk, cyclin dependent kinase; GC, gastrocnemius; si, Small Interfering; Fbxo, F-box protein; Trim, tripartite motif; Mfn, mitofusin; p-Drp1, phosphorylated Dynamin related protein 1; Myog, myogenin; Mstn, myostatin; DEX, dexamethasone; NC, Negative control.

Article Snippet: Viability of C2C12 myoblasts after GW8510 treatment was performed using CCK-8 (Shanghai Yeasen Biotechnology Co., Ltd.; cat. no. 40203ES60).

Techniques: Expressing, Western Blot, Transfection, Control, Negative Control

Journal: Life Metabolism

Article Title: Baf60c in skeletal muscle regulates adipose tissue thermogenesis via Musclin-mediated endocrine signaling

doi: 10.1093/lifemeta/loaf015

Figure Lengend Snippet: Baf60c regulates Musclin expression in a cell-autonomous manner. (a) Heatmap depicting the expression of upregulated and downregulated genes in C2C12-derived myotubes with Baf60c knockdown (cutoff: P < 0.05 and |average change| > 1) compared to the control myotubes. (b) The Baf60c (left) and Musclin (right) mRNA expression in Baf60c siRNA-mediated knockdown and scramble siRNA-treated control myotubes ( n = 3 per group). (c) The Baf60c (left) and Musclin (right) mRNA expression in Baf60c OE C2C12 cells ( n = 3 per group). (d and e) The Baf60c (d) and Musclin (e) mRNA expression in C2C12-differentiated myotubes after starvation for 0, 8, 16, 24, and 36 h ( n = 3 per group). During starvation, cells were treated by DMEM supplemented with low glucose (1 mmol/L) and 0.1% BSA. *** P < 0.001, by one-way ANOVA with multiple comparisons. (f) The Baf60c (left) and Musclin (right) mRNA expression in myotubes cultured at different temperatures ( n = 3 per group). Data represent mean ± SD. *** P < 0.001, by two-tailed unpaired Student’s t -test, unless otherwise noted.

Article Snippet: C2C12 myoblasts were purchased from the American Type Culture Collection (ATCC) and cultured in DMEM containing 10% FBS.

Techniques: Expressing, Derivative Assay, Knockdown, Control, Cell Culture, Two Tailed Test

Journal: Life Metabolism

Article Title: Baf60c in skeletal muscle regulates adipose tissue thermogenesis via Musclin-mediated endocrine signaling

doi: 10.1093/lifemeta/loaf015

Figure Lengend Snippet: Baf60c regulates Musclin expression through interacting with Mef2c. (a) HOMER motif analysis on ATAC-seq data of quadriceps muscles from 3-month-old Bc flox/flox and BcMKO mice. (b) Physical interaction of Baf60c with Mef2a and Mef2c in HEK293T cells. HEK293T cells were transiently transfected with Myc-tagged Baf60c (Baf60c) and Flag/HA-tagged Mef2a or Mef2c, followed by IP with anti-HA agarose beads and immunoblotting. (c) qPCR analysis of the Baf60c (left), Mef2c (middle), and Musclin (right) gene expression in Mef2c overexpressed and control myotubes with either Baf60c knockdown or not ( n = 3). (d) The Baf60c expression in Baf60c knockout C2C12 cells (left panel), and qPCR analysis of the Mef2c (middle panel) and Musclin (right panel) gene expression in Baf60c knockout and control myotubes with either Mef2c overexpression or not ( n = 3). Data represent mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, no significance, by one-way ANOVA with multiple comparisons.

Article Snippet: C2C12 myoblasts were purchased from the American Type Culture Collection (ATCC) and cultured in DMEM containing 10% FBS.

Techniques: Expressing, Muscles, Transfection, Western Blot, Gene Expression, Control, Knockdown, Knock-Out, Over Expression

Journal: Molecular Metabolism

Article Title: Training-induced plasma miR-29a-3p is secreted by skeletal muscle and contributes to metabolic adaptations to resistance exercise in mice

doi: 10.1016/j.molmet.2025.102173

Figure Lengend Snippet: In vitro study of the impact of EPS in miRNA secretion and the modulatory effect of miR-29a-3p inhibition in training-associated miRNAs in mouse myotubes. A . Schematic representation of electrostimulation protocol on C2C12 cells and isolation of miRNAs from EVs and cells. Created with BioRender. B-D. Relative expression levels of miR-30a-5p, let-7f-5p, and miR-29a-3p in cells and EVs at 0, 3, and 20 h after electrostimulation in non-electrostimulated (C, n = 4) and electrostimulated (EPS, n = 4) cells. E-G. Relative expression of cluster 1 miRNAs ( E ), let-7i-5p ( F ), and miR-143-3p ( G ) in C2C12 myotubes where miR-29a-3p expression was inhibited by 70% (miR29dKD, n = 4) and in control myotubes (Control, n = 4). H–I. Relative expression of miR-30a-5p and miR-143-3p in non-electrostimulated (C, n = 4) and electrostimulated (EPS, n = 4) miR-29a-3p dKD cells at 0, 3, and 20 h after electrostimulation. Statistical significance was determined using unpaired, two-tailed Student’s t -test and p -values are indicated in the figures. Data are represented as mean ± SEM. Each dot represents the mean technical duplicate of each cell replicate.

Article Snippet: The mouse skeletal muscle myoblast cell line C2C12 were obtained from the American Type Culture Collection (ATCC CRL-1772).

Techniques: In Vitro, Inhibition, Isolation, Expressing, Control, Two Tailed Test