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: 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