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: It permits that all cells express the miRNA avoiding the influence of untransfected cells. pCMVmiR-GFP vector (Origene) containing miR-26a, -134a, or -431 gene were used to transfect C2C12 cells.

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: It permits that all cells express the miRNA avoiding the influence of untransfected cells. pCMVmiR-GFP vector (Origene) containing miR-26a, -134a, or -431 gene were used to transfect C2C12 cells.

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

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: In brief, C2C12 cells were transfected with miRNA mimics and 100 pg/mL of pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega) containing the target sequence or a control sequence (primers for cloning are listed in ).

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: In brief, C2C12 cells were transfected with miRNA mimics and 100 pg/mL of pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega) containing the target sequence or a control sequence (primers for cloning are listed in ).

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

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: In brief, C2C12 cells were transfected with miRNA mimics and 100 pg/mL of pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega) containing the target sequence or a control sequence (primers for cloning are listed in ).

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: In brief, C2C12 cells were transfected with miRNA mimics and 100 pg/mL of pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega) containing the target sequence or a control sequence (primers for cloning are listed in ).

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

Journal: Bioactive Materials

Article Title: A sandwich-like nanofibrous scaffold with macrophage phenotype transformation and myogenic differentiation for skeletal muscle regeneration

doi: 10.1016/j.bioactmat.2025.05.008

Figure Lengend Snippet: Aligned and random PCL fibers regulate cell morphology and myogenic differentiation. A, D) STROM images of cell morphology, B, E) radar maps of cytoskeleton orientation, C, F) nuclear morphology and aspect ratio of C2C12 cells and NIH3T3 cells cultured on random and aligned PCL fibrous scaffolds for 3 days. The white bidirectional arrow indicates the direction of the cytoskeleton. (n = 30; ∗∗∗∗ p < 0.0001). G) Representative STROM images of IF staining of MHC and H) corresponding relative expression levels of C2C12 cells after 5 days of myogenic-induced culture on random and aligned PCL scaffolds (n = 3; ∗∗ p < 0.01). Heat map of expression of I) cell morphology-related genes and J) myogenic-related genes in C2C12 cells cultured on random and aligned PCL scaffolds for 3 days.

Article Snippet: A, D) STROM images of cell morphology, B, E) radar maps of cytoskeleton orientation, C, F) nuclear morphology and aspect ratio of C2C12 cells and NIH3T3 cells cultured on random and aligned PCL fibrous scaffolds for 3 days.

Techniques: Cell Culture, Staining, Expressing

Journal: Bioactive Materials

Article Title: A sandwich-like nanofibrous scaffold with macrophage phenotype transformation and myogenic differentiation for skeletal muscle regeneration

doi: 10.1016/j.bioactmat.2025.05.008

Figure Lengend Snippet: The adhesion and myogenic differentiation of C2C12 cells on the surfaces of aligned PCL scaffold, aligned PG scaffold, and the sandwich-like PGH scaffold. A) Representative STROM images of IF staining of Vinculin, B) corresponding mean fluorescence intensity, and C) aspect ratio of nucleus after 3 days of culture on P, PG, and PGH scaffolds (n = 3; ∗∗ p < 0.01, ∗∗∗ p < 0.001). D) Representative STROM images of IF staining of MHC, E) corresponding mean fluorescence intensity, and F) aspect ratio of nucleus after 3 days of myogenic-induced culture on P, PG, and PGH scaffolds (n = 3; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001). G) qPCR analysis genes expression levels associated with myogenic differentiation in C2C12 cells after culturing on P, PG, and PGH scaffolds for 3 days (n = 3; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001). H) Schematic diagram of PGH scaffold regulating C2C12 cell behavior.

Article Snippet: A, D) STROM images of cell morphology, B, E) radar maps of cytoskeleton orientation, C, F) nuclear morphology and aspect ratio of C2C12 cells and NIH3T3 cells cultured on random and aligned PCL fibrous scaffolds for 3 days.

Techniques: Staining, Fluorescence, Expressing

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: To induce muscle atrophy in vitro , C2C12 cells were stimulated with 10 μ M dexamethasone (TargetMol Chemicals Inc.; cat. no. T1076) or 20 ng/ml TNFα (Novoprotein Scientific Inc.; cat. no. CF09) for 24 h at 37°C.

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: To induce muscle atrophy in vitro , C2C12 cells were stimulated with 10 μ M dexamethasone (TargetMol Chemicals Inc.; cat. no. T1076) or 20 ng/ml TNFα (Novoprotein Scientific Inc.; cat. no. CF09) for 24 h at 37°C.

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: To induce muscle atrophy in vitro , C2C12 cells were stimulated with 10 μ M dexamethasone (TargetMol Chemicals Inc.; cat. no. T1076) or 20 ng/ml TNFα (Novoprotein Scientific Inc.; cat. no. CF09) for 24 h at 37°C.

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: To induce muscle atrophy in vitro , C2C12 cells were stimulated with 10 μ M dexamethasone (TargetMol Chemicals Inc.; cat. no. T1076) or 20 ng/ml TNFα (Novoprotein Scientific Inc.; cat. no. CF09) for 24 h at 37°C.

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: To induce muscle atrophy in vitro , C2C12 cells were stimulated with 10 μ M dexamethasone (TargetMol Chemicals Inc.; cat. no. T1076) or 20 ng/ml TNFα (Novoprotein Scientific Inc.; cat. no. CF09) for 24 h at 37°C.

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: To induce muscle atrophy in vitro , C2C12 cells were stimulated with 10 μ M dexamethasone (TargetMol Chemicals Inc.; cat. no. T1076) or 20 ng/ml TNFα (Novoprotein Scientific Inc.; cat. no. CF09) for 24 h at 37°C.

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: The activity of SOD and CK in serum of muscle atrophy mice and in C2C12 cells was measured using a SOD (cat. no. A001-3-2) and CK assay kit (both Nanjing Jiancheng Bioengineering Institute; cat. no. A032-1-1) according to the manufacturer's instructions.

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: The activity of SOD and CK in serum of muscle atrophy mice and in C2C12 cells was measured using a SOD (cat. no. A001-3-2) and CK assay kit (both Nanjing Jiancheng Bioengineering Institute; cat. no. A032-1-1) according to the manufacturer's instructions.

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: The activity of SOD and CK in serum of muscle atrophy mice and in C2C12 cells was measured using a SOD (cat. no. A001-3-2) and CK assay kit (both Nanjing Jiancheng Bioengineering Institute; cat. no. A032-1-1) according to the manufacturer's instructions.

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: The activity of SOD and CK in serum of muscle atrophy mice and in C2C12 cells was measured using a SOD (cat. no. A001-3-2) and CK assay kit (both Nanjing Jiancheng Bioengineering Institute; cat. no. A032-1-1) according to the manufacturer's instructions.

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: The activity of SOD and CK in serum of muscle atrophy mice and in C2C12 cells was measured using a SOD (cat. no. A001-3-2) and CK assay kit (both Nanjing Jiancheng Bioengineering Institute; cat. no. A032-1-1) according to the manufacturer's instructions.

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: The activity of SOD and CK in serum of muscle atrophy mice and in C2C12 cells was measured using a SOD (cat. no. A001-3-2) and CK assay kit (both Nanjing Jiancheng Bioengineering Institute; cat. no. A032-1-1) according to the manufacturer's instructions.

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

Journal: Bioactive Materials

Article Title: A sandwich-like nanofibrous scaffold with macrophage phenotype transformation and myogenic differentiation for skeletal muscle regeneration

doi: 10.1016/j.bioactmat.2025.05.008

Figure Lengend Snippet: Aligned and random PCL fibers regulate cell morphology and myogenic differentiation. A, D) STROM images of cell morphology, B, E) radar maps of cytoskeleton orientation, C, F) nuclear morphology and aspect ratio of C2C12 cells and NIH3T3 cells cultured on random and aligned PCL fibrous scaffolds for 3 days. The white bidirectional arrow indicates the direction of the cytoskeleton. (n = 30; ∗∗∗∗ p < 0.0001). G) Representative STROM images of IF staining of MHC and H) corresponding relative expression levels of C2C12 cells after 5 days of myogenic-induced culture on random and aligned PCL scaffolds (n = 3; ∗∗ p < 0.01). Heat map of expression of I) cell morphology-related genes and J) myogenic-related genes in C2C12 cells cultured on random and aligned PCL scaffolds for 3 days.

Article Snippet: C2C12 cells were obtained from Warner Bio (Wuhan) Co., Ltd. (China) and cultured in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin-streptomycin (MP Biomedicals, USA).

Techniques: Cell Culture, Staining, Expressing

Journal: Bioactive Materials

Article Title: A sandwich-like nanofibrous scaffold with macrophage phenotype transformation and myogenic differentiation for skeletal muscle regeneration

doi: 10.1016/j.bioactmat.2025.05.008

Figure Lengend Snippet: The adhesion and myogenic differentiation of C2C12 cells on the surfaces of aligned PCL scaffold, aligned PG scaffold, and the sandwich-like PGH scaffold. A) Representative STROM images of IF staining of Vinculin, B) corresponding mean fluorescence intensity, and C) aspect ratio of nucleus after 3 days of culture on P, PG, and PGH scaffolds (n = 3; ∗∗ p < 0.01, ∗∗∗ p < 0.001). D) Representative STROM images of IF staining of MHC, E) corresponding mean fluorescence intensity, and F) aspect ratio of nucleus after 3 days of myogenic-induced culture on P, PG, and PGH scaffolds (n = 3; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001). G) qPCR analysis genes expression levels associated with myogenic differentiation in C2C12 cells after culturing on P, PG, and PGH scaffolds for 3 days (n = 3; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001). H) Schematic diagram of PGH scaffold regulating C2C12 cell behavior.

Article Snippet: C2C12 cells were obtained from Warner Bio (Wuhan) Co., Ltd. (China) and cultured in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin-streptomycin (MP Biomedicals, USA).

Techniques: Staining, Fluorescence, Expressing

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: To induce muscle atrophy in vitro , C2C12 cells were stimulated with 10 μ M dexamethasone (TargetMol Chemicals Inc.; cat. no. T1076) or 20 ng/ml TNFα (Novoprotein Scientific Inc.; cat. no. CF09) for 24 h at 37°C.

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: To induce muscle atrophy in vitro , C2C12 cells were stimulated with 10 μ M dexamethasone (TargetMol Chemicals Inc.; cat. no. T1076) or 20 ng/ml TNFα (Novoprotein Scientific Inc.; cat. no. CF09) for 24 h at 37°C.

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: To induce muscle atrophy in vitro , C2C12 cells were stimulated with 10 μ M dexamethasone (TargetMol Chemicals Inc.; cat. no. T1076) or 20 ng/ml TNFα (Novoprotein Scientific Inc.; cat. no. CF09) for 24 h at 37°C.

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: To induce muscle atrophy in vitro , C2C12 cells were stimulated with 10 μ M dexamethasone (TargetMol Chemicals Inc.; cat. no. T1076) or 20 ng/ml TNFα (Novoprotein Scientific Inc.; cat. no. CF09) for 24 h at 37°C.

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: To induce muscle atrophy in vitro , C2C12 cells were stimulated with 10 μ M dexamethasone (TargetMol Chemicals Inc.; cat. no. T1076) or 20 ng/ml TNFα (Novoprotein Scientific Inc.; cat. no. CF09) for 24 h at 37°C.

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: To induce muscle atrophy in vitro , C2C12 cells were stimulated with 10 μ M dexamethasone (TargetMol Chemicals Inc.; cat. no. T1076) or 20 ng/ml TNFα (Novoprotein Scientific Inc.; cat. no. CF09) for 24 h at 37°C.

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