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: 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: 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: HEK293T and C2C12 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, 11995065, Gibco) supplemented with 10% fetal bovine serum (FBS, SE100-011, VisTech), streptomycin (50 μg/mL), and penicillin (50 U/mL, 15140122, Gibco).

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: HEK293T and C2C12 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, 11995065, Gibco) supplemented with 10% fetal bovine serum (FBS, SE100-011, VisTech), streptomycin (50 μg/mL), and penicillin (50 U/mL, 15140122, Gibco).

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 C2C12 cell line was cultured in DMEM (Dulbecco’s Modified Eagle Medium, 11965092, Gibco, Thermo Fisher Scientific, Inc., MA, USA).

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

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: For tissue, EVs and C2C12 cells analysis equal quantities of RNA (5 ng/μL) were used. cDNA was synthesized using the miRCURY LNA RT kit (QIAGEN, Hilden, Germany) [ ].

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