cell culture c2c12 murine skeletal muscle myoblasts  (ATCC)

Journal: Human Genetics and Genomics Advances

Article Title: Using a modular massively parallel reporter assay to discover context-dependent regulatory activity in type 2 diabetes-linked noncoding regions

doi: 10.1016/j.xhgg.2026.100606

Figure Lengend Snippet: HNF1 transcription factor motifs contribute to enhancer activity near selected T2D-associated variants (A) For 47 HNF1-motif-overlapping fragments with significant INS promoter-bias effects on activity, we designed three versions: original (motif intact), deleted (motif removed and sequence adjusted), and shuffled (dinucleotide-shuffled motif). When the tested variant was adjacent to the motif, we synthesized both reference and alternative alleles for each version. For variants directly overlapping the motif, we generated only one deletion and one shuffled fragment. (B) We synthesized four fragments corresponding to the variant rs1635852, which overlaps an HNF1 motif at a high-information-content position. The T2D risk allele (T) disrupts this motif, while the non-risk allele (C) matches the consensus. (C) Shuffling the motif significantly decreased enhancer activity compared to intact fragments with either the risk T (Wilcoxon rank-sum test p = 0.016) or non-risk C ( p = 0.008) allele. Motif deletion also significantly decreased enhancer activity compared to the non-risk C allele ( p = 0.016). (D) For the variant rs11819995, located 11 bp upstream of an HNF1 motif, we synthesized six fragments. (E) Deletion of the motif significantly decreased enhancer activity for both the reference (C, non-risk) and alternative (T, risk) alleles ( p = 0.008 for both alleles). Shuffling the motif likewise reduced activity for both alleles ( p = 0.008 for the reference allele and p = 0.056 for the alternative allele). (F) To assess context-specific effects, we cloned these fragments into MPRA vectors with the SCP1 or skeletal-muscle-specific MYBPC2 promoter and delivered all three to LHCN-M2 human skeletal muscle myotubes ( n = 6). (G) When paired with the INS promoter, the shuffled rs11819995-containing fragment showed increased activity relative to the original fragment ( p = 0.015); however, none of the fragments containing rs11819995 functioned as enhancers in LHCN-M2 myotubes, regardless of promoter context. Overall, their activity is highest when paired with the skeletal-muscle-specific promoter.

Article Snippet: We obtained INS-1 832/13 rat insulinoma cells from Dr. Christopher Newgard (Sarah W. Stedman Nutrition and Metabolism Center, Duke University, Durham, NC) and LHCN-M2 human skeletal muscle myoblasts from Evercyte.

Techniques: Activity Assay, Sequencing, Variant Assay, Synthesized, Generated, Clone Assay

Journal: Bioactive Materials

Article Title: Anisotropic mechanotransductive tissue constructs via brush-assisted bioprinting of microfiber-reinforced composite bioinks

doi: 10.1016/j.bioactmat.2025.12.017

Figure Lengend Snippet: Comparative analysis of conventional and brush-assisted bioprinting on cellular behavior. (a) Schematic illustration of shear stress distribution in normal versus brush-assisted printing. (b) Overview of the brush-assisted printing setup. (c) SEM images of collagen fibrils and fluorescence images showing cell viability (live/dead) and cytoskeletal organization (DAPI/phalloidin) of C2C12 myoblasts. Quantification of (d) cell viability post-printing (n = 4), (e) cell metabolic activity (MTT assay, in situ /day 3/day 7, n = 4), (f) nuclei aspect ratio (n = 180), (g) orientation factor (n = 3), and (h) F-actin–positive area at day 3 (n = 10). (i) Comparing normal and brush-assisted printing the mechanotransduction pathways activated by shear stress and collagen alignment. (j) Heatmap of relative gene expression ( YAP, TAZ, AKT1, PIEZO1, PI3K, and CAPN2 ) after 7 days of culture (n = 4). (k) Agarose gel electrophoresis of PCR products from cells cultured on normal versus brush-printed scaffolds for 7 days. (l) Schematic illustration of blocking mechano-sensing ion channel with GsMTx-4. (m) Relative gene expression levels associated with mechanosensing channel and ca 2+ pathway (n = 5), (n) Hippo pathway (n = 5), (o) PI3K-AKT pathway (n = 5). Student's t-test was applied for two-group comparisons, and one-way ANOVA with Tukey's HSD post-hoc test was used for multiple comparisons. Data are presented as mean ± standard deviation (SD). Statistical significance was set at p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.001 (∗∗∗). Abbreviation: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (cell metabolic activity assay); F-actin, Filamentous actin; PI3K , Phosphoinositide 3-kinase; CAPN2 , Calcium-activated neutral protease 2.

Article Snippet: H9C2 cardiomyoblasts (Korean Cell Line Bank, Seoul, Korea) and C2C12 murine skeletal muscle myoblasts (CRL-1772, ATCC, Manassas, USA) were cultured in high-glucose DMEM (Welgene, Korea) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin–streptomycin (PS).

Techniques: Shear, Fluorescence, Activity Assay, MTT Assay, In Situ, Gene Expression, Agarose Gel Electrophoresis, Cell Culture, Blocking Assay, Standard Deviation, Metabolic Assay

Journal: Bioactive Materials

Article Title: Anisotropic mechanotransductive tissue constructs via brush-assisted bioprinting of microfiber-reinforced composite bioinks

doi: 10.1016/j.bioactmat.2025.12.017

Figure Lengend Snippet: Physical and biological evaluation of bioconstructs reinforced with straight (CSP) and coiled (CCP) PCL fibers. (a) Schematic illustration and optical images of flow tests with Col (collagen-only), CSP, and CCP bioinks, in which droplets were tilted at 90° for 10 min. (b) Quantification of droplet outflow (n = 10). Rheological measurements of bioinks: (c) storage modulus (G′) from frequency sweep (n = 3), (d) G′ and G″ from temperature sweep (n = 3), and (e) G′ and G″ under cyclic stress loading (10 and 200 Pa) showing viscoelastic recovery (n = 3). (f) SEM images of CCP constructs highlighting coiled PCL fiber and aligned collagen fibrils. (g) Optical image, live/dead staining, DAPI/phalloidin staining, and orientation factor analysis of C2C12 cells and PCL microfibers. Quantification of (h) cell viability (live/dead, n = 4), (i) F-actin–positive area (n = 20), and (j) metabolic activity (MTT assay, in situ /day 3/day 7, n = 4). Student's t-test was applied for two-group comparisons, and one-way ANOVA with Tukey's HSD post-hoc test was used for multiple comparisons. Data are presented as mean ± standard deviation (SD). Statistical significance was set at p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.001 (∗∗∗).

Article Snippet: H9C2 cardiomyoblasts (Korean Cell Line Bank, Seoul, Korea) and C2C12 murine skeletal muscle myoblasts (CRL-1772, ATCC, Manassas, USA) were cultured in high-glucose DMEM (Welgene, Korea) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin–streptomycin (PS).

Techniques: Construct, Staining, Activity Assay, MTT Assay, In Situ, Standard Deviation

Journal: Bioactive Materials

Article Title: Anisotropic mechanotransductive tissue constructs via brush-assisted bioprinting of microfiber-reinforced composite bioinks

doi: 10.1016/j.bioactmat.2025.12.017

Figure Lengend Snippet: In vitro myogenic differentiation of C2C12 cells cultured on Col, CSP and CCP scaffolds. (a) Live/dead staining at in situ , DAPI/phalloidin staining at day 3, and DAPI/ MHC staining at day 14. (b) Quantification of cell viability from live/dead assays (n = 4). (c) Nuclei orientation factor after 7 days of culture (n = 4). (d) Nuclei aspect ratio (n = 20). (e) F-actin positive area (n = 4). (f) Quantification of MHC fusion index (left, n = 5) and MHC maturation rate (right, n = 5). (g) Relative gene expression analysis and (h) agarose gel electrophoresis regarding mechanotransduction-related genes ( CAPN2, PIEZO1, RhoA, YAP, and TAZ ) (n = 4). (i) Western blot analysis of PIEZO1 . (j) Schematic illustrating differentiation progression and major genes involved at each stage. (k) Heatmap and (l) agarose gel electrophoresis of PCR products showing relative expression of myogenic markers ( MYF5, MYOD1, MYOG, MHC, MYH2, and MYH4 ) after 21 days of culture (n = 4). (m) Western blot analysis of MHC . Student's t-test was applied for two-group comparisons, and one-way ANOVA with Tukey's HSD post-hoc test was used for multiple comparisons. Data are presented as mean ± standard deviation (SD). Statistical significance was set at p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.001 (∗∗∗). Abbreviation: MYF5 , Myogenic factor 5; MYOD1 , Myogenic differentiation 1; MYOG , Myogenin; MHC , Myosin heavy chain; MYH2 , Myosin heavy chain 2; MYH4 , Myosin heavy chain 4.

Article Snippet: H9C2 cardiomyoblasts (Korean Cell Line Bank, Seoul, Korea) and C2C12 murine skeletal muscle myoblasts (CRL-1772, ATCC, Manassas, USA) were cultured in high-glucose DMEM (Welgene, Korea) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin–streptomycin (PS).

Techniques: In Vitro, Cell Characterization, Cell Culture, Staining, In Situ, Gene Expression, Agarose Gel Electrophoresis, Western Blot, Expressing, Standard Deviation

Journal: Physiological Reports

Article Title: Synthetic estrogen and progestin effects on the myogenic program following damage in C2C12 murine myoblasts

doi: 10.14814/phy2.70886

Figure Lengend Snippet: (a) Representative images of C2C12 myotubes 6 days after differentiation in vehicle (VEH), OC1, OC2, OC3, and OC4 formulations. Myotubes were stained with desmin (green) and DAPI (blue). (b, d, f) Myotube diameter (um) of vehicle, EE, progestin, and OC formulations after 6 days of differentiation. (c, e, g) Myonuclear index of vehicle, EE, progestin and OC formulations after 6 days of differentiation. Values are presented as median lines and interquartile range (boxes) ± maximum and minimum values (whiskers), with + representing the mean. Statistical comparisons were performed using a one‐way ANOVA. p ‐value indicates a significant difference from vehicle (VEH).

Article Snippet: Murine C2C12 skeletal muscle myoblasts from American Type Culture Collection (Cedarlane) were cultured in uncoated 150 mm tissue culture dishes in 5% CO 2 at 37°C.

Techniques: Staining

Journal: Autophagy Reports

Article Title: Autophagy selectively clears ER in TNF-α-induced muscle atrophy

doi: 10.1080/27694127.2026.2649064

Figure Lengend Snippet: Dynamic SILAC reveals distinct sets of proteins affected in their turnover upon TNF-α-induced muscle atrophy in C2C12 cells. (A) Experimental workflow: C2C12 myoblasts were differentiated for seven days in light medium (K0 = Lysine 0 [ 12 C 6 , 14 N 2 ]; R0 = Arginine 0 [ 12 C 6 , 14 N 4 ]) into myotubes (= t0). Medium was then replaced with heavy medium (K8 = Lysine 8 [ 13 C 6 , 15 N 2 ]; R10 = Arginine 10 [ 13 C 6 , 15 N 4 ]) for 24 or 72 h (t24, t72), with or without TNF-α to induce atrophy. Proteolytic inhibitors (BafA1: Bafilomycin A1 inhibiting autophagy; Lac: Lactacystin inhibiting the UPS) were administered for 3 h prior to cell harvest, and cells were analyzed using LC-MS/MS. n = 3 replicates. (B) Myotube diameter was measured in differentiated C2C12 myotubes under homeostatic conditions at 24 h (H24; day 8 of differentiation) and 72 h (H72; day 10), as well as following TNF-α-induced atrophy at corresponding time points (A24 and A72). Measurements were performed using ImageJ on 209 individual myotubes per condition, quantified from 15-20 randomly selected images. Statistical significance was determined using ordinary one-way ANOVA with multiple comparisons (GraphPad Prism). Images used were acquired from OPP-stained samples from . (C) Protein clustering based on total intensity dynamics of non-inhibitor treated homeostatic and atrophying cells: Proteins were categorized into four clusters according to their normalized total intensity values (light + heavy) at t0, t24, and t72. Percentages of protein counts were rounded up. (D) Proteins of each cluster of total intensities were subjected to GO term enrichment (biological processes) analysis.

Article Snippet: WT C2C12 mouse skeletal muscle myoblasts (ATCC, CRL-1772) and ssGFP-KDEL C2C12 cells (kindly provided and originally described by Buonomo et al . [ ]) were grown adherently and undifferentiated in DMEM (Sigma-Aldrich, D5796) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Gibco, 10500-064) and 1% Pen/Strep (P/S) (Sigma-Aldrich, P4333).

Techniques: Multiplex sample analysis, Liquid Chromatography with Mass Spectroscopy, Staining

Journal: Autophagy Reports

Article Title: Autophagy selectively clears ER in TNF-α-induced muscle atrophy

doi: 10.1080/27694127.2026.2649064

Figure Lengend Snippet: TNF-α-induced atrophy leads to acute translation inhibition. (A) OPP (O-propargyl-puromcyin) labeling of differentiated myotubes (homeostatic at 24 h (H24) and 72 h (H27)) and TNF-α-induced atrophying C2C12 cells (24 h (A24) and 72 h (A72)) was used to measure protein synthesis rates at time points t24 and t72. Arrows indicate puncta representing newly translated proteins. Asterisks indicate nuclei in differentiated, multinucleated myotubes. (B) Cycloheximide (CHX) was employed for 90 min as a control to inhibit protein synthesis. Puncta were manually counted across 20 images per condition, each containing over 200 cells. (C) Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test. Scale bar: 60 µm.

Article Snippet: WT C2C12 mouse skeletal muscle myoblasts (ATCC, CRL-1772) and ssGFP-KDEL C2C12 cells (kindly provided and originally described by Buonomo et al . [ ]) were grown adherently and undifferentiated in DMEM (Sigma-Aldrich, D5796) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Gibco, 10500-064) and 1% Pen/Strep (P/S) (Sigma-Aldrich, P4333).

Techniques: Inhibition, Labeling, Control

Journal: Autophagy Reports

Article Title: Autophagy selectively clears ER in TNF-α-induced muscle atrophy

doi: 10.1080/27694127.2026.2649064

Figure Lengend Snippet: Increased autophagic turnover in TNF-α-induced atrophying C2C12 cells. (A) Western blot analysis of LC3B-II and p62 in C2C12 myotubes under homeostatic (H24, H72) and atrophic (A24, A72) conditions, with and without Bafilomycin (BafA1) treatment at time points 24 h and 72 h. Vehicle-treated (DMSO) cells serve as control. (B) LC3B-II and p62 intensities were normalized against Vinculin. n = 3 replicates. ns = not significant. (C-E) Flow cytometry-based assay measuring autophagic turnover by staining membrane-bound LC3-II with a fluorescently labeled antibody (C) Representative histograms display the shift in LC3-II signal following 3 h BafilomycinA1 (BafA1) treatment (D) Elevated LC3-II signal intensity (gMFI, geometric mean fluorescence intensity) in atrophying (A24, A72) cells compared to homeostatic cells (H24, H72) in dimethylsulfoxide (DMSO)-treated samples. n = 4 replicates. ns = not significant. (E) Autophagic turnover was calculated by determining the difference in LC3-II signal intensity (gMFI) between BafA1-treated and DMSO-treated cells over a 3 h incubation period. n = 4 replicates. (F) Accumulated proteins upon BafilomycinA1 (BafA1) treatment in ANOVA comparisons of atrophic (A24, A72) vs. homeostatic conditions (H24, H72) in the light channel (log 2 FC > 0 adj. p -value < 0.05). Left: Venn diagram numbers depict significantly regulated proteins per comparison. Right: Red dots in volcano plot depict autophagy machinery components.

Article Snippet: WT C2C12 mouse skeletal muscle myoblasts (ATCC, CRL-1772) and ssGFP-KDEL C2C12 cells (kindly provided and originally described by Buonomo et al . [ ]) were grown adherently and undifferentiated in DMEM (Sigma-Aldrich, D5796) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Gibco, 10500-064) and 1% Pen/Strep (P/S) (Sigma-Aldrich, P4333).

Techniques: Western Blot, Control, Flow Cytometry, Staining, Membrane, Labeling, Fluorescence, Incubation, Comparison

Journal: Autophagy Reports

Article Title: Autophagy selectively clears ER in TNF-α-induced muscle atrophy

doi: 10.1080/27694127.2026.2649064

Figure Lengend Snippet: During TNF-a-induced atrophy, canonical autophagy does not mediate myofibrillar protein degradation, whereas ER-phagy emerges as the predominant form of selective autophagy. (A) Immunocytochemical staining of C2C12 (atrophying) myotubes for p62/Sqstm1 and myosin heavy chain (MyHC) highlighting the M band region of the sarcomere with corresponding line plots displaying fluorescence intensity profiles for each channel along the length of the sarcomere. Scale bar 20 µm. (B) Accumulated myofibrillar proteins upon BafilomycinA1 (+B) and Lactacystin (+L) treatment in ANOVA comparisons of homeostatic (H) and atrophying (A) C2C12 cells (light channel; log 2 FC > 0, adj. p -value < 0.05). White: not significantly accumulated; grey: not significant. (C) ANOVA of differentially regulated proteins (log 2 FC > 0; adj. p -value < 0.05). Left: Newly synthesized proteins in atrophy (A24 vs. H24 and A72 vs. H72) and accumulating (Light channel) selective autophagy receptors ± BafilomycinA1 (+B) treatment in atrophy (A24 vs. H24 and A72 vs. H72). Right: Accumulating ER components ± BafilomycinA1 (+B in atrophy (A24 vs. H24 and A72 vs. H24). White: not significantly accumulated; gray: not significant.

Article Snippet: WT C2C12 mouse skeletal muscle myoblasts (ATCC, CRL-1772) and ssGFP-KDEL C2C12 cells (kindly provided and originally described by Buonomo et al . [ ]) were grown adherently and undifferentiated in DMEM (Sigma-Aldrich, D5796) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Gibco, 10500-064) and 1% Pen/Strep (P/S) (Sigma-Aldrich, P4333).

Techniques: Staining, Fluorescence, Synthesized

Journal: Autophagy Reports

Article Title: Autophagy selectively clears ER in TNF-α-induced muscle atrophy

doi: 10.1080/27694127.2026.2649064

Figure Lengend Snippet: Schematic model of proteostatic remodeling in TNF-α-induced muscle atrophy over time (24 h to 72 h): In C2C12 myotubes, TNF-α triggers selective remodeling of proteostasis, translation (Ribosomal and mitochondrial ribosomal proteins), ER stress, autophagic flux and autophagic cargo. Arrows represent changes observed under TNF-α-induced atrophy relative to the corresponding baseline control.

Article Snippet: WT C2C12 mouse skeletal muscle myoblasts (ATCC, CRL-1772) and ssGFP-KDEL C2C12 cells (kindly provided and originally described by Buonomo et al . [ ]) were grown adherently and undifferentiated in DMEM (Sigma-Aldrich, D5796) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Gibco, 10500-064) and 1% Pen/Strep (P/S) (Sigma-Aldrich, P4333).

Techniques: Control

Journal: Preventive Nutrition and Food Science

Article Title: Puerarin-Rich Pueraria lobata Extract Attenuates H 2 O 2 - and Dexamethasone-Induced Atrophy in C2C12 Myoblasts and Myotubes

doi: 10.3746/pnf.2025.307

Figure Lengend Snippet: Cytotoxicity of Pueraria lobata extract powder (PEP) and its protective effect against H 2 O 2 -induced oxidative stress in C2C12 myoblasts. Cell viability was measured using an MTT assay. (A) C2C12 myoblasts were treated with the indicated concentrations of PEP for 24 h. Data are presented as a percentage of the untreated control group. (B) Cells were pretreated with PEP for 1 h, followed by cotreatment with 100 µM H 2 O 2 for a further 24 h. Data are presented as a percentage of the H 2 O 2 -only treated group. All values are presented as the mean±standard error of the mean (n=4). *** P <0.001 vs. the untreated normal control group; # P <0.05 vs. the H 2 O 2 -only treated group.

Article Snippet: Mouse skeletal muscle C2C12 myoblasts (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco) at 37°C in a humidified incubator with 5% CO 2 .

Techniques: MTT Assay, Control

Journal: Preventive Nutrition and Food Science

Article Title: Puerarin-Rich Pueraria lobata Extract Attenuates H 2 O 2 - and Dexamethasone-Induced Atrophy in C2C12 Myoblasts and Myotubes

doi: 10.3746/pnf.2025.307

Figure Lengend Snippet: Pueraria lobata extract powder (PEP) inhibits H 2 O 2 -induced apoptosis in C2C12 myoblasts. Cells were pretreated with the indicated concentrations of PEP for 1 h, followed by cotreatment with 100 µM H 2 O 2 for a further 24 h. (A) Apoptosis was quantified by measuring DNA fragmentation using a commercial ELISA. Data are presented as a percentage of the H 2 O 2 -only treated group. (B) Representative fluorescence microscopy images of cells stained with Hoechst 33258, showing nuclear morphology. Apoptotic nuclei appear brightly stained and condensed (magnification, ×200). All values are presented as the mean±standard error of the mean (n=6). *** P <0.001 vs. the untreated normal control group (CON); ## P <0.01 and ### P <0.001 vs. the H 2 O 2 -only treated group.

Article Snippet: Mouse skeletal muscle C2C12 myoblasts (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco) at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Enzyme-linked Immunosorbent Assay, Fluorescence, Microscopy, Staining, Control

Journal: Preventive Nutrition and Food Science

Article Title: Puerarin-Rich Pueraria lobata Extract Attenuates H 2 O 2 - and Dexamethasone-Induced Atrophy in C2C12 Myoblasts and Myotubes

doi: 10.3746/pnf.2025.307

Figure Lengend Snippet: Pueraria lobata extract powder (PEP) ameliorates dexamethasone (DEX)-induced atrophy in C2C12 myotubes. C2C12 myotubes were treated with 5 µM DEX in the presence or absence of the indicated concentrations of PEP for 24 h. (A) Cell viability was measured using an MTT assay and is expressed as a percentage relative to the control group. (B) Myotube diameter was quantified from immunofluorescence images and is expressed in micrometers (µm). (C) Representative immunofluorescence images of myotubes stained for myosin heavy chain (red) and nuclei with DAPI (blue). Scale bar=100 µm (magnification ×200). Data are presented as the mean±standard error of the mean (n=3). ** P <0.01 and *** P <0.001 vs. the control group (CON); # P <0.05, ## P <0.01, and ### P <0.001 vs. the DEX-only treated group.

Article Snippet: Mouse skeletal muscle C2C12 myoblasts (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco) at 37°C in a humidified incubator with 5% CO 2 .

Techniques: MTT Assay, Control, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: PTBP1 inhibition reprograms myogenesis to rescue impaired muscle regeneration in mdx mice through correcting E2A splicing

doi: 10.1038/s41467-026-70669-9

Figure Lengend Snippet: a H&E staining of triceps brachii (TB) muscle section from individuals without DMD (unaffected controls) and DMD patients (DMD). b , c Serum creatine kinase (CK) and lactate dehydrogenase (LDH) concentration. d Immunofluorescence co-staining for embryonic myosin heavy chain (eMyHC, representing nascent myofiber) and MyOD (representing proliferating myoblast) of triceps brachii (TB) section. e Immunofluorescence co-staining for eMyHC and myosin heavy chain (MHC, representing mature myofiber) of TB section. f Relative mRNA expression level of paired box protein 7 ( PAX7 ), myogenic factor 5 ( MYF5 ) , MyOD1, myogenin ( MYOG ) , myogenic regulatory factor 4 ( MRF4 ), transcription factor E2-alpha ( E2A ) and its two subtypes (E12 and E47). g Schematic representation of basic helix-loop-helix (bHLH) domain amino acid sequence and E12/E47 variant amino acid sequence. h Determination of mRNA expression level of E12 and E47 using 8% polyacrylamide gel electrophoresis (PAGE) (up) and quantification (down). E47(18b) was specifically cut by PstI restriction enzyme. i Schematic representation of myogenic regulatory factors (MRFs) expressed in different phases of muscle regeneration. j Myotube formation in C2C12 myoblast myogenic differentiation process for continuous 4 days. k Relative mRNA expression level of PAX7 and MRFs during C2C12 myoblast differentiation. l Determination of the mRNA expression level of E12 and E47 in proliferated myoblast (PRO) and differentiated myoblast undergo continuous 4 days myogenic differentiation (DIF) using 8% PAGE (up) and quantification (down). m Protein expression level of E12, E47 and E2A (left) and associated quantification (right). n Protein expression level of E2A, MyOD, and MyOG (left) and associated quantification (right) in C2C12 myoblast for continuous 4 days myogenic differentiation. n = 6 independent biological replicates ( a – f ). n = 3 independent biological replicates ( h , j – n ). Scale bars: 100 μm ( a , d , e ), 200 μm ( j ). Unpaired two-tailed Student’s t tests were performed in ( b , c , f , m ) (E47 and E12); one-way ANOVA plus Tukey’s post hoc tests were performed in ( k , n ) (MyOD and MyOG); two-way ANOVA plus Tukey’s post hoc tests were performed in ( h , l , m (E2A) and n (E2A)). All data show the means ± SD. Source data are provided as a Source Data file.

Article Snippet: Murine skeletal muscle myoblast C2C12 (CRL-1772) line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Staining, Concentration Assay, Immunofluorescence, Expressing, Sequencing, Variant Assay, Polyacrylamide Gel Electrophoresis, Cell Characterization, Two Tailed Test

Journal: Nature Communications

Article Title: PTBP1 inhibition reprograms myogenesis to rescue impaired muscle regeneration in mdx mice through correcting E2A splicing

doi: 10.1038/s41467-026-70669-9

Figure Lengend Snippet: a Schematic representation of E12 and E47 mRNA was generated from mutually exclusive alternative splicing (MEAS) of exon 18 in E2A pre-mRNA. b Specific overexpression or siRNA plasmid targeting E12 or E47 mRNA were design based on their transcript variant isoform and variant domain. c – i E12 or E47 were specifically knockdown (E12-KD or E47-KD) or overexpressed (E12-OE or E47-OE) in C2C12 myoblast proliferation phase. d , g Relative mRNA expression level of E12 and E47 . e , h Protein expression level of E2A and MyOD (left) and associated quantification (right). f , i H&E staining of myoblast. Scale bar: 100 μm. j – p E12 or E47 were specifically knockdown or overexpressed in the C2C12 myoblast differentiation phase. k , n Relative mRNA expression level of E12 and E47 . l , o Protein expression level of E2A and MyOG (left) and associated quantification (right). m , p Immunofluorescence staining of myotube (left) and associated evaluation for the numbers of multinucleated myotubes normalize to the negative control (NC) group (right). Scale bar: 100 μm. n = 3 independent biological replicates ( c – p ). One-way ANOVA plus Tukey’s post hoc tests were performed in ( e (MyOD), h (MyOD), l (MyOG), o (MyOG), m and n ); two-way ANOVA plus Tukey’s post hoc tests were performed in (d, g , k , n , e (E2A), h (E2A), l (E2A), o (E2A)). All data show the means ± SD. Source data are provided as a Source Data file.

Article Snippet: Murine skeletal muscle myoblast C2C12 (CRL-1772) line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Generated, Alternative Splicing, Over Expression, Plasmid Preparation, Variant Assay, Knockdown, Expressing, Staining, Immunofluorescence, Negative Control

Journal: Nature Communications

Article Title: PTBP1 inhibition reprograms myogenesis to rescue impaired muscle regeneration in mdx mice through correcting E2A splicing

doi: 10.1038/s41467-026-70669-9

Figure Lengend Snippet: a Screening of key differentially expressed genes (DEGs) in heterogeneous nuclear ribonucleoprotein ( HNRNP ) and dead-box RNA helicase ( DDX ) family members between proliferating myoblast and differentiated myotube ( GSE79263 ) using dealt TPM > 124 (10% maximum changed TPM) and change percentage > 40% (left). The detailed expression level of key DEGs (right). Red points indicate upregulation in myotubes, blue points indicate downregulation. b Relative expression of key DEGs in skeletal muscle of DMD patients compared with unaffected controls based on the GEO database ( GSE38417 ). Red indicates upregulation in DMD, blue indicates downregulation. c Relative mRNA expression of the key DEGs in skeletal muscle biopsies from unaffected controls and DMD patients. d Protein expression level of PTBP1 in skeletal muscle biopsies (up) and associated quantification (below). e Immunofluorescence co-staining for eMyHC and MyOD of triceps brachii (TB) section. Scale bar: 100 μm. f , g Relative mRNA expression of Ptbp1 , protein expression level of PTBP1, E2A (left) and associated quantification (right) in C2C12 myoblast for continuous 4 days differentiation. h Immunofluorescence co-staining for eMyHC and PTBP1 in C2C12 myoblast for continuous 4 days differentiation. Scale bar: 40 μm. i According to ( h ) quant i fication of PTBP1 protein level in continuous 4 days differentiation (up). Quantification of PTBP1 in myotube, the relative PTBP1 protein level in myotube normalized to the surrounding undifferentiated myoblast (down). j , k Immunofluorescence co-staining for PTBP1 and MyOD ( j ), PTBP1 and MyOG ( k ) in undifferentiated myoblast and myotube. Scale bar: 20 μm. l According to ( j , k ) quantification of eMyHC, PTBP1, MyOD, MyOG protein level in proliferating myoblasts and myotube, eMyHC and MyOG protein level were normalized to myotube, PTBP1 and MyOD protein level was normalized to myoblast. n = 6 independent biological replicates ( c – e ). n = 3 independent biological replicates ( h - l ). Unpaired two-tailed Student’s t tests were performed in ( c , d , l ); one-way ANOVA plus Tukey’s post hoc tests were performed in ( f, g (PTBP1)), and ( i ) two-way ANOVA plus Tukey’s post hoc tests were performed in ( g (E2A)). All data show the means ± SD. Source data are provided as a Source Data file.

Article Snippet: Murine skeletal muscle myoblast C2C12 (CRL-1772) line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Immunofluorescence, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: PTBP1 inhibition reprograms myogenesis to rescue impaired muscle regeneration in mdx mice through correcting E2A splicing

doi: 10.1038/s41467-026-70669-9

Figure Lengend Snippet: a – e PTBP1 knockdown (PTBP1-KD) or overexpression (PTBP1-OE) in C2C12 myoblast proliferation phase. b , d Protein expression level of PTBP1, E2A and MyOD (left) and associated quantification (right). c , e H&E staining of myoblast. Scale bar: 100 μm. f , j PTBP1were knockdown or overexpressed in C2C12 myoblast differentiation phase. g , i Protein expression level of PTBP1, E2A and MyOG (left) and associated quantification (right). h , j Immunofluorescence staining of myotube (left) and associated evaluation for the numbers of multinucleated myotubes normalize to the negative control (NC) group (right). Scale bar: 200 μm. k Protein expression level of PTBP1, E2A and MyOG under PTBP1 overexpression combined with E12 overexpression or not (left) and associated quantification (right). l Immunofluorescence staining of myotube (left) and associated evaluation for the numbers of multinucleated myotubes normalize to the NC group (right). Scale bar: 200 μm. m Protein expression level of PTBP1, E2A and MyOG under PTBP1 knockdown combined with E12 knockdown or not (left) and associated quantification (right). n Immunofluorescence staining of myotube (left) and associated evaluation for the numbers of multinucleated myotubes normalize to the NC group (right). Scale bar: 200 μm. n = 3 independent biological replicates ( b – e , g , n ). Unpaired two-tailed Student’s t tests were performed in ( h , j ) and all proteins except E2A in ( b , d , g , and i ); one-way ANOVA plus Tukey’s post hoc tests were performed in ( l , n ) and PTBP1, MyOG proteins in ( k , m ); two-way ANOVA plus Tukey’s post hoc tests were performed in E2A protein in b , d , g , i , k , and m . All data show the means ± SD. Source data are provided as a Source Data file.

Article Snippet: Murine skeletal muscle myoblast C2C12 (CRL-1772) line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knockdown, Over Expression, Expressing, Staining, Immunofluorescence, Negative Control, Two Tailed Test

Journal: Nature Communications

Article Title: PTBP1 inhibition reprograms myogenesis to rescue impaired muscle regeneration in mdx mice through correcting E2A splicing

doi: 10.1038/s41467-026-70669-9

Figure Lengend Snippet: a Key potential differentially expressed genes (DEGs) in Usp family members were screened between C2C12 proliferated myoblast vs differentiated myotube, Dealt TPM > 12.1 (10% maximum changed TPM) and change percentage > 50% was set as standard for key potential target gene, the red points represented upregulated genes in myogenic differentiation, while the blue points represented downregulation. b Relative mRNA expression level of the key potential DEGs in wildtype (WT) mice and mdx mice. c Relative mRNA expression level of USP9X in skeletal muscle of unaffected controls and DMD patients using biopsy specimen. d Protein expression level of USP9X in C2C12 myoblast for continuous 4 days myogenic differentiation. e Immunofluorescence of USP9X protein level in nuclei of myoblast and myotube. Scale bar: 20 μm. f – l Degrasyn promotes PTBP1 protein degradation and muscle regeneration in mdx mice, mdx was intraperitoneally injected with 15 mg/kg/d, 30 mg/kg/d Degrasyn (Deg-) or 5 mg/kg/d prednisolone (Pred). f Protein expression level of USP9X, PTBP1, MyOD, and MyOG (left) and associated quantification (right). g H&E staining of GAS, QF, TA, and DIA sections. Scale bar: 100 μm. h Immunofluorescence co-staining for DAPI, eMyHC, and MHC of GAS section, scale bar: 100 μm. i Hanging behavior and time (up) and associated quantification (down). j , k Serum CK and LDH concentration. l Walking gait (left) and associated quantification (right). n = 3 independent biological replicates ( a , d , e ). n = 6 independent biological replicates ( b , c , f – l ). Unpaired two-tailed Student’s t tests were performed in ( b and c ); one-way ANOVA plus Tukey’s post hoc tests were performed in ( d , f , i – l ). All data show the means ± SD. Source data are provided as a Source Data file.

Article Snippet: Murine skeletal muscle myoblast C2C12 (CRL-1772) line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Cell Characterization, Expressing, Immunofluorescence, Injection, Staining, Concentration Assay, Two Tailed Test