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