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: 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

Journal: bioRxiv

Article Title: Deuterated Polyunsaturated Fatty Acids Alleviate In Vitro Skeletal Muscle Dysfunction Induced by Oxidative Stress

doi: 10.64898/2025.12.29.696779

Figure Lengend Snippet: (A): To assess the effect of H-PUFAs and D-PUFAs during differentiation, confluent C2C12 myoblasts were induced to differentiate for 72 hours in DM supplemented with a 25 µM dose of individual H-PUFAs or D-PUFAs. (B-D): Total DAPI + cell count (B) , average myonuclei per myotube (C) , and average myotube diameter during differentiation (D) were quantified using ImageJ. (E): To assess the effect of H-PUFAs and D-PUFAs on mature myotubes, confluent C2C12 myoblasts were induced to differentiate for 72 hours in DM prior to supplementation with a 25 µM dose of individual H-PUFAs or D-PUFAs for 72 hours. (F-H): Total DAPI + cell count (F) , average myonuclei per myotube (G) , and myotube diameter (H) were quantified using ImageJ. For both experiments, myotubes were fixed by 4% PFA and visualized after immunofluorescent staining for sarcomeric myosin (MF20c, colored green) and myogenin (F5Dc, colored red). Cell nuclei were counterstained with DAPI (blue). Scale bar is 200 µm. *P<0.05 for difference compared to vehicle, #P<0.05 for difference between H-PUFA and D-PUFA.

Article Snippet: The C2C12 murine skeletal muscle cell line (ATCC, CRL-1772) was cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco, 11995-065) containing 10% fetal bovine serum (FBS, Corning, 35-010-CV) and antibiotics (penicillin 100 U/mL, streptomycin 100 μg/mL, Gibco, 15140-122) at 37 °C in humid air with 5% CO 2 .

Techniques: Cell Counting, Staining

Journal: bioRxiv

Article Title: Deuterated Polyunsaturated Fatty Acids Alleviate In Vitro Skeletal Muscle Dysfunction Induced by Oxidative Stress

doi: 10.64898/2025.12.29.696779

Figure Lengend Snippet: ( A): To assess the effect of H 2 O 2 on mature myotubes, confluent C2C12 myoblasts were induced to differentiate for 48 h in DM and then pre-treated with a 25 µM dose of individual H-PUFA or D-PUFA for a further 24 h. Subsquently, myotubes continued to grow in fresh DM with 2 mM H 2 O 2 and a fresh 25 µM of H-PUFAs or D-PUFAs for 48 hours. Myotubes were fixed in 4% PFA and visualized after immunofluorescent staining for sarcomeric myosin (MF20c, colored green) and myogenin (F5Dc, colored red). Cell nuclei were counterstained with DAPI (blue). Scale bar is 200 µm. (B-D): Total DAPI + cell count ( B ), average myonuclei per myotube ( C ), and myotube area ( D ) were quantified using ImageJ. ( E): Bodipy C11 staining of C2C12 cells receiving H-PUFA or D-PUFA in the presence of H 2 O 2 . Oxidized lipids are in green and reduced lipids are in red. *P<0.05 for difference of PUFAs compared to H 2 O 2 , #P<0.05 for difference between H-PUFA and D-PUFA.

Article Snippet: The C2C12 murine skeletal muscle cell line (ATCC, CRL-1772) was cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco, 11995-065) containing 10% fetal bovine serum (FBS, Corning, 35-010-CV) and antibiotics (penicillin 100 U/mL, streptomycin 100 μg/mL, Gibco, 15140-122) at 37 °C in humid air with 5% CO 2 .

Techniques: Staining, Cell Counting

Journal: bioRxiv

Article Title: Deuterated Polyunsaturated Fatty Acids Alleviate In Vitro Skeletal Muscle Dysfunction Induced by Oxidative Stress

doi: 10.64898/2025.12.29.696779

Figure Lengend Snippet: (A) To assess the effect of erastin during differentiation, proliferating C2C12 myoblasts were pretreated with a 25 µM dose of individual D-PUFAs including D-ARA, D-EPA, D-DPA, or D-DHA for 24 hours. Confluent myoblasts were then induced to differentiate in DM containing ferroptosis inducer erastin (10 µM) and fresh D-PUFAs (25 µM) for 72 hours. (B-D): Total DAPI + cell count ( B ), average myonuclei per myotube ( C ), and average myotube diameter ( D ) were measured using ImageJ. (E): To assess the effect of erastin on mature myotubes, confluent C2C12 cells were induced to differentiate in DM for 48 hours and then pretreated with individual D-PUFAs for 24 hours. Subsquently, myotubes continued to grow in fresh DM containing erastin (10 µM) and fresh D-PUFAs for 72 hours. (F-H): Total DAPI + cell count ( F ), average myonuclei per myotube ( G ), and myotube diameter ( H ) were quantified using ImageJ. Myotubes were fixed and visualized after immunofluorescent staining for sarcomeric myosin (MF20c, colored green) and myogenin (F5Dc, colored red). Cell nuclei were counterstained with DAPI (blue). Scale bar is 200 µm. *P<0.05 for difference during differentiation, #P<0.05 for difference post differentiation.

Article Snippet: The C2C12 murine skeletal muscle cell line (ATCC, CRL-1772) was cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco, 11995-065) containing 10% fetal bovine serum (FBS, Corning, 35-010-CV) and antibiotics (penicillin 100 U/mL, streptomycin 100 μg/mL, Gibco, 15140-122) at 37 °C in humid air with 5% CO 2 .

Techniques: Cell Counting, Staining

Journal: bioRxiv

Article Title: Deuterated Polyunsaturated Fatty Acids Alleviate In Vitro Skeletal Muscle Dysfunction Induced by Oxidative Stress

doi: 10.64898/2025.12.29.696779

Figure Lengend Snippet: (A): To assess the effect of D-PUFA dose during differentiation, proliferating C2C12 myoblasts were pretreated with an increasing dose of D-ARA, D-ALA, or D-LA (0, 25, 50, 100 µM) for 24 hours. Confluent myoblasts were then induced to differentiate for 72 h in DM containing erastin (10 µM) and fresh D-ARA, D-ALA, or D-LA at respective concentrations. Myotubes were fixed and visualized after immunofluorescent staining for sarcomeric myosin (MF20c, colored green) and myogenin (F5Dc, colored red). Cell nuclei were counterstained with DAPI (blue). Scale bar is 200 µm. (B-G): Myotube diameter ( B, D, & F ) and average nuclei per myotube ( C, E, & G ) was measured in cultures receiving treatment with D-ARA ( B-C ), D-ALA ( D-E ), and D-LA ( F-G ). (H): Neutral lipid stained by BODIPY 493/503 in the dose response experiment. Representative images shown in (A) and (H ) were taken from the same fields of view. *P<0.05 for difference compared to cells receiving erastin alone.

Article Snippet: The C2C12 murine skeletal muscle cell line (ATCC, CRL-1772) was cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco, 11995-065) containing 10% fetal bovine serum (FBS, Corning, 35-010-CV) and antibiotics (penicillin 100 U/mL, streptomycin 100 μg/mL, Gibco, 15140-122) at 37 °C in humid air with 5% CO 2 .

Techniques: Staining

Journal: bioRxiv

Article Title: Deuterated Polyunsaturated Fatty Acids Alleviate In Vitro Skeletal Muscle Dysfunction Induced by Oxidative Stress

doi: 10.64898/2025.12.29.696779

Figure Lengend Snippet: At 3 days post-differentiation, mature C2C12 myotubes were co-treated with erastin (10 µM) and D-PUFAs (25 µM) in fresh DM for 24 hours. Cell mRNA expression of Scl7a11 ( A ), Hmox1 ( B ), Fbxo32 ( C ), and Il6 ( D ) were assessed using RT-qPCR as described in methods. Gene expression was normalized to Tbp . *P<0.05 for effect of D-PUFAs compared to vehicle, #P<0.05 for effect of erastin.

Article Snippet: The C2C12 murine skeletal muscle cell line (ATCC, CRL-1772) was cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco, 11995-065) containing 10% fetal bovine serum (FBS, Corning, 35-010-CV) and antibiotics (penicillin 100 U/mL, streptomycin 100 μg/mL, Gibco, 15140-122) at 37 °C in humid air with 5% CO 2 .

Techniques: Expressing, Quantitative RT-PCR, Gene Expression