mouse c2c12 myoblasts  (ATCC)

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Elevated Circulating Ceramides 18:0 and 24:1 as a Risk Factor for Sarcopenia: In Vitro, Animal, and Clinical Evidence

doi: 10.1002/jcsm.70310

Figure Lengend Snippet: C18:0 ceramide impairs in vitro myogenic differentiation. (a) Mouse C2C12 myoblasts were differentiated into myotubes using 2% horse serum in the presence of the indicated concentrations of C18:0 ceramide for 3 days. Myotubes were immunostained with an anti‐MyHC antibody, and nuclei were counterstained with DAPI. Quantitative analyses per field are shown ( n = 4). (b) Western blot and (c) quantitative reverse transcription polymerase chain reaction analyses were performed to assess the expression levels of myogenic markers myogenin and MyHC following 3‐day treatment with 0.1 μM C18:0 ceramide during differentiation ( n = 3). (d) Myoblast migration was evaluated using a Boyden chamber assay, and (e) cell viability was measured with a CCK‐8 assay after treatment with the indicated concentrations of C18:0 ceramide for 6 and 24 h, respectively ( n = 5). (f) The inhibitory effects of C18:0 ceramide on myogenic differentiation were similarly confirmed using primary myoblasts under the same experimental conditions as described in (a). Scale bars: 100 μm (a), 50 μm (d), 100 μm (f). MyHC, myosin heavy chain; DAPI, 4′,6‐diamidino‐2‐phenylindole; OD, optical density. * p < 0.05 vs. untreated control.

Article Snippet: Mouse C2C12 myoblasts (American Type Culture Collection, Manassas, Virginia, United States) were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% foetal bovine serum (FBS), 20 mM HEPES, 2 mM L‐glutamine, 100 U/mL penicillin and 0.1 mg/mL streptomycin (all from Life Technologies, Carlsbad, California, United States).

Techniques: In Vitro, Cell Characterization, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Expressing, Migration, Boyden Chamber Assay, CCK-8 Assay, Control

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Elevated Circulating Ceramides 18:0 and 24:1 as a Risk Factor for Sarcopenia: In Vitro, Animal, and Clinical Evidence

doi: 10.1002/jcsm.70310

Figure Lengend Snippet: The inhibitory effects of C18:0 ceramide on myogenesis are mediated by increased intracellular ROS generation. (a) Mouse C2C12 myoblasts were differentiated into myotubes using 2% horse serum in the presence of the indicated concentrations of C18:0 ceramide for 3 days. Intracellular ROS levels were assessed using the fluorescent probe chloromethyl‐2′,7′‐dichlorofluorescein diacetate (CM‐H 2 DCFDA) ( n = 5). (b,c) C2C12 cells were differentiated into myotubes with 2% horse serum in the presence or absence of 0.1 μM C18:0 ceramide and/or 1 mM NAC for 3 days. (b) Intracellular ROS levels were measured using H 2 DCFDA ( n = 3). (c) Myotubes were stained with anti‐MyHC antibody, and nuclei were counterstained with DAPI. Quantitative analyses of myogenic parameters per field are shown ( n = 3). (d) Expression level of MyHC was assessed by western blotting under the same conditions ( n = 3). (e,f) C2C12 cells were treated with 0.1 μM C18:0 ceramide and/or 1 mM NAC for 1 day ( n = 3). (e) SA‐β‐gal‐positive cells (%) per field were quantified, and representative images of SA‐β‐gal staining are shown. SA‐β‐gal‐positive cells appear blue. (f) Western blot analyses were performed to assess p21 and p16 expression levels. β‐Tubulin served as a loading control. Scale bars: 100 μm (b,c), 20 μm (e). ROS, reactive oxygen species; NAC, N‐acetyl cysteine; MyHC, myosin heavy chain; DAPI, 4′,6‐diamidino‐2‐phenylindole; SA‐β‐gal, senescence‐associated β‐galactosidase. * p < 0.05 vs. untreated control; # p < 0.05 vs. 0.1 μM C18:0 ceramide.

Article Snippet: Mouse C2C12 myoblasts (American Type Culture Collection, Manassas, Virginia, United States) were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% foetal bovine serum (FBS), 20 mM HEPES, 2 mM L‐glutamine, 100 U/mL penicillin and 0.1 mg/mL streptomycin (all from Life Technologies, Carlsbad, California, United States).

Techniques: Staining, Expressing, Western Blot, Control

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Elevated Circulating Ceramides 18:0 and 24:1 as a Risk Factor for Sarcopenia: In Vitro, Animal, and Clinical Evidence

doi: 10.1002/jcsm.70310

Figure Lengend Snippet: C24:1 ceramide impairs in vitro myogenic differentiation. (a) Mouse C2C12 myoblasts were differentiated into myotubes using 2% horse serum in the presence of the indicated concentrations of C24:1 ceramide for 3 days. Myotubes were immunostained with an anti‐MyHC antibody, and nuclei were counterstained with DAPI. Quantitative analyses per field are shown ( n = 4). (b) Western blot and (c) quantitative reverse transcription polymerase chain reaction analyses were conducted to evaluate the expression of myogenic markers, myogenin and MyHC, following 3‐day treatment with 0.1 μM C24:1 ceramide during differentiation ( n = 3). (d) Myoblast migration was assessed using a Boyden chamber assay, and (e) cell viability was measured using a CCK‐8 assay after treatment with the indicated concentrations of C24:1 ceramide for 6 and 24 h, respectively ( n = 5). (f) The inhibitory effects of C24:1 ceramide on myogenic differentiation were also confirmed in primary myoblasts under the same experimental conditions as described in (a). Scale bars: 100 μm (a), 50 μm (d), 100 μm (f). MyHC, myosin heavy chain; DAPI, 4′,6‐diamidino‐2‐phenylindole; OD, optical density. * p < 0.05 vs. untreated control.

Article Snippet: Mouse C2C12 myoblasts (American Type Culture Collection, Manassas, Virginia, United States) were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% foetal bovine serum (FBS), 20 mM HEPES, 2 mM L‐glutamine, 100 U/mL penicillin and 0.1 mg/mL streptomycin (all from Life Technologies, Carlsbad, California, United States).

Techniques: In Vitro, Cell Characterization, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Expressing, Migration, Boyden Chamber Assay, CCK-8 Assay, Control

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Elevated Circulating Ceramides 18:0 and 24:1 as a Risk Factor for Sarcopenia: In Vitro, Animal, and Clinical Evidence

doi: 10.1002/jcsm.70310

Figure Lengend Snippet: The inhibitory effects of C24:1 ceramide on myogenesis are mediated by increased intracellular ROS generation. (a) Mouse C2C12 myoblasts were differentiated into myotubes using 2% horse serum in the presence of the indicated concentrations of C24:1 ceramide for 3 days. Intracellular ROS levels were assessed using the fluorescent probe chloromethyl‐2′,7′‐dichlorofluorescein diacetate (CM‐H 2 DCFDA) ( n = 5). (b,c) C2C12 cells were differentiated into myotubes with 2% horse serum in the presence or absence of 0.1 μM C24:1 ceramide and/or 1 mM NAC for 3 days. (b) Intracellular ROS levels were measured using H 2 DCFDA ( n = 3). (c) Myotubes were stained with anti‐MyHC antibody, and nuclei were counterstained with DAPI. Quantitative analyses of myogenic parameters per field are shown ( n = 3). (d) Expression level of MyHC was assessed by western blotting under the same conditions ( n = 3). (e,f) C2C12 cells were treated with 0.1 μM C24:1 ceramide and/or 1 mM NAC for 1 day ( n = 3). (e) SA‐β‐gal‐positive cells (%) per field were quantified, and representative images of SA‐β‐gal staining are shown. SA‐β‐gal‐positive cells appear blue. (f) Western blot analyses were performed to assess p21 and p16 expression levels. β‐Tubulin served as a loading control. Scale bars: 100 μm (b and c), 20 μm (e). ROS, reactive oxygen species; NAC, N‐acetyl cysteine; MyHC, myosin heavy chain; DAPI, 4′,6‐diamidino‐2‐phenylindole; SA‐β‐gal, senescence‐associated β‐galactosidase. * p < 0.05 vs. untreated control; # p < 0.05 vs. 0.1 μM C24:1 ceramide.

Article Snippet: Mouse C2C12 myoblasts (American Type Culture Collection, Manassas, Virginia, United States) were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% foetal bovine serum (FBS), 20 mM HEPES, 2 mM L‐glutamine, 100 U/mL penicillin and 0.1 mg/mL streptomycin (all from Life Technologies, Carlsbad, California, United States).

Techniques: Staining, Expressing, Western Blot, Control

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Elevated Circulating Ceramides 18:0 and 24:1 as a Risk Factor for Sarcopenia: In Vitro, Animal, and Clinical Evidence

doi: 10.1002/jcsm.70310

Figure Lengend Snippet: Ceramide‐induced oxidative stress suppresses ITGB1 signalling and promotes skeletal muscle atrophy in vitro. (a,b) Differentiated C2C12 myotubes were treated with vehicle (Veh), C18:0 ceramide or C24:1 ceramide in the presence or absence of NAC. (a) Western blot analyses were performed to assess ITGB1 protein expression ( n = 3). (b) Myotubes were immunostained with an anti‐ITGB1 antibody, and nuclei were counterstained with DAPI ( n = 5). (c) Western blot analyses were conducted to evaluate the activation status of ITGB1‐associated signalling pathways in myotubes treated with C18:0 or C24:1 ceramide in the presence or absence of the ITGB1‐activating antibody TS2/16. (d) Myotubes were immunostained with an anti‐FoxO1 antibody to assess nuclear localisation following treatment with C18:0 or C24:1 ceramide with or without TS2/16. Nuclei were counterstained with DAPI ( n = 5). (e) mRNA expression levels of MuRF1 and Atrogin‐1 were evaluated by quantitative reverse transcription polymerase chain reaction ( n = 3). (f) Myotube width, myotube area per myotube and myotube size distribution were quantified to assess myotube atrophy following ceramide treatment with or without TS2/16 ( n = 5). Scale bars: 100 μm (b,d). An asterisk (*) indicates a statistically significant difference among groups (b–f).

Article Snippet: Mouse C2C12 myoblasts (American Type Culture Collection, Manassas, Virginia, United States) were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% foetal bovine serum (FBS), 20 mM HEPES, 2 mM L‐glutamine, 100 U/mL penicillin and 0.1 mg/mL streptomycin (all from Life Technologies, Carlsbad, California, United States).

Techniques: In Vitro, Western Blot, Expressing, Activation Assay, Reverse Transcription, Polymerase Chain Reaction

Journal: Research

Article Title: Doping-Engineered Proangiogenic Nanozymes Orchestrate Ischemic Tissue Regeneration via Cytoprotection and Revascularization

doi: 10.34133/research.1260

Figure Lengend Snippet: Cu-doped Prussian blue (CuPB) nanozymes protect C2C12 myoblasts and H9c2 cardiomyocytes from H 2 O 2 -induced oxidative injury. (A and B) Representative fluorescence images and quantification of intracellular reactive oxygen species (ROS) in H 2 O 2 -injured C2C12 cells after Prussian blue (PB) or CuPB treatment, detected using the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe. Scale bar: 50 μm. n = 5. (C and D) Representative terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining images and quantification of apoptotic C2C12 cells following H 2 O 2 injury with PB or CuPB treatment. Scale bar: 50 μm. n = 5. (E) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of apoptosis-related genes ( Bcl2 , Caspase3 , Caspase9 , and Bax ) in C2C12 cells after different treatments. n = 3. (F and G) Representative fluorescence images and quantification of intracellular ROS in H 2 O 2 -injured H9c2 cells after PB or CuPB treatment, detected using the DCFH-DA probe. Scale bar: 50 μm. n = 5. (H and I) Representative TUNEL staining images and quantification of apoptotic H9c2 cells following H 2 O 2 injury with PB or CuPB treatment. Scale bar: 50 μm. n = 5. (J) qRT-PCR analysis of apoptosis-related gene expression ( Bcl2 , Caspase3 , Caspase9 , and Bax ) in H9c2 cells after different treatments. n = 5.

Article Snippet: The rat cardiomyocyte cell line (H9c2) was obtained from Procell Life Science & Technology Co., Ltd. (China), and the mouse myoblast cell line (C2C12) was purchased from Beijing Zhongyuan Heju Biotechnology Co., Ltd., the authorized American Type Culture Collection distributor in China (CRL1772).

Techniques: Fluorescence, End Labeling, TUNEL Assay, Staining, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Gene Expression

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