Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: ZBED6 Knockout Prevents Ageing‐ and Dexamethasone‐Induced Muscle Atrophy via Dkk3 in Pig and Mice

doi: 10.1002/jcsm.13829

Figure Lengend Snippet: Zbed6 controls Dkk3 transcription in ageing‐induced muscle atrophy. (A) Representative H&E staining (top), Sirius (middle), fibre‐type staining (bottom) and quantification (right) of histological cross sections in GM tissues of Zbed6 −/− and controls of 18 months old mice. Myosin heavy chain type I and IIa (red), IIb (green) and DAPI (blue). Representative images are shown. Scale bar = 200 μm. n = 3; (B) Dkk3, Fbxo32 and Murf1 mRNA expression levels in mice described in A. n = 3. (C) Representative western blotting and quantification of Dkk3, Fbxo32 and Murf1 in mice described in A. GAPDH served as internal control. n = 3. (D) Representative western blotting of Zbed6 in WT and Zbed6‐KO C2C12 cells ( n = 3) and representative images for fibre diameter and area of myotubes (myogenic differentiation takes 72 h). Red indicated myosin heavy chain (MyHC) immunofluorescent staining and DAPI (blue). Scale bars = 100 μm. (E) Dkk3, Fbxo32 and Murf1 mRNA expression levels decreased after Zbed6 depleted. n = 4. GAPDH served as internal control. Data are expressed as mean ± SEM; * p < 0.05, ** p < 0.01.

Article Snippet: The C2C12 cell line (ATCC) was cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 4.5 g/L glucose (11955065, Gibco, United States), 10% foetal bovine serum (FBS) (10099141C, Gibco, United States) and 1% penicillin–streptomycin solution (P1400, Solarbio, Beijing, China).

Techniques: Staining, Expressing, Western Blot, Control

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: ZBED6 Knockout Prevents Ageing‐ and Dexamethasone‐Induced Muscle Atrophy via Dkk3 in Pig and Mice

doi: 10.1002/jcsm.13829

Figure Lengend Snippet: Overexpression of Dkk3 downregulation Zbed6 deficiency‐mediated against muscle atrophy in vivo and vitro. (A) Schematic representation of mouse model. AAV9‐myo2A‐GFP and AAV‐myo2A‐Dkk3 were injected in TA muscle of the left and right legs of the same mice (3‐month‐old) in Zbed6 f/f and Zbed6 −/− mice for one month, and the following experimental setup ( n = 3 mice of Zbed6 f/f ; n = 4 mice of Zbed6 −/− ) (left). Representative western blotting and quantification of Flag‐Dkk3 (right). (B) Dkk3, Fbxo32 and Murf1 mRNA expression levels in mice described in A. (C) Representative H&E staining (left), average CSA (upper right) and TA muscle weight in mice described in A. Scale bars = 200 μm. (D) Schematic representation of cultured myotubes using 2% horse serum; the differentiation was induced to form myotubes for 72 h and then infected by adenovirus‐encoding Dkk3 or control vector for 48 h. (E) Representative images and quantification (left) for fibre diameter and area of myotubes infected for 120 hours in C2C12 described in D. Red indicated myosin heavy chain (MyHC) immunofluorescent staining and DAPI (blue). Scale bars = 100 μm. Dkk3, Murf1 and Fbxo32 mRNA expression level (right). n = 3. (F) Representative western blotting and quantification of Zbed6, Dkk3, Fbxo32, Murf1, phosphorylated protein levels of FoxO3 and total FoxO3 in C2C12 described in D. GAPDH served as internal control. Data are expressed as mean ± SEM; * p < 0.05, ** p < 0.01.

Article Snippet: The C2C12 cell line (ATCC) was cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 4.5 g/L glucose (11955065, Gibco, United States), 10% foetal bovine serum (FBS) (10099141C, Gibco, United States) and 1% penicillin–streptomycin solution (P1400, Solarbio, Beijing, China).

Techniques: Over Expression, In Vivo, Injection, Western Blot, Expressing, Staining, Cell Culture, Infection, Control, Plasmid Preparation

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: ZBED6 Knockout Prevents Ageing‐ and Dexamethasone‐Induced Muscle Atrophy via Dkk3 in Pig and Mice

doi: 10.1002/jcsm.13829

Figure Lengend Snippet: Zbed6 depletion protects dexamethasone‐induced myotubes and muscles atrophy. (A) Schematic representation of Dex injection, representative H&E staining and quantification of histological cross sections derived from Zbed6 −/− and controls (3‐month‐old) TA muscles, and mice was injected with or without 15 mg/kg/day of Dex for 10 days. Scale bars = 100 μm. n = 6. (B) mRNA expression levels of Zbed6, Dkk3, Murf1 and Fbxo32 described in A. n = 6. (C) Representative western blotting and quantification of Zbed6, Dkk3, Murf1 and Fbxo32 in mice described in A. n = 3. (D) Representative images (top) and quantification (bottom) for fibre diameter and area of myotubes. WT and Zbed6‐KO C2C12 myotubes with or without Dex; the myotubes were treated with 100 μM Dex for 24 h. Red indicated myosin heavy chain (MyHC) immunofluorescent staining and DAPI (blue). Scale bars = 100 μm. n = 3. (E, F) mRNA and protein expression levels of Zbed6, Dkk3, Murf1 and Fbxo32 described in D. n = 3. GAPDH served as internal control. Data are expressed as mean ± SEM; * p < 0.05, ** p < 0.01.

Article Snippet: The C2C12 cell line (ATCC) was cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 4.5 g/L glucose (11955065, Gibco, United States), 10% foetal bovine serum (FBS) (10099141C, Gibco, United States) and 1% penicillin–streptomycin solution (P1400, Solarbio, Beijing, China).

Techniques: Muscles, Injection, Staining, Derivative Assay, Expressing, Western Blot, Control

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: ZBED6 Knockout Prevents Ageing‐ and Dexamethasone‐Induced Muscle Atrophy via Dkk3 in Pig and Mice

doi: 10.1002/jcsm.13829

Figure Lengend Snippet: Overexpression of Zbed6 accelerates muscle atrophy in vitro and vivo . (A) Representative images (left) and quantification (right) of myotubes. Primary myotubes overexpressing Zbed6 by adenovirus‐encoding Zbed6 or control vector, 24 h after infection, the myotubes infection with Si‐Dkk3 or negative‐control (NC). Red indicated myosin heavy chain (MyhC) immunofluorescent staining and DAPI (blue). Scale bars = 200 μm. n = 3. (B) Zbed6, Dkk3, Murf1 and Fbxo32 mRNA expression level in C2C12 described in A. n = 4. (C) Schematic representation of virus and Dex injection. AAV9‐myo2A‐GFP and AAV‐myo2A‐Zbed6 were injected in TA muscle of the left and right legs of the same mice (3‐month‐old). One month later, the mice were intraperitoneally injected with or without 15 mg/kg/day of dexamethasone for 10 days. n = 3. (D) Representative H&E staining (left) and quantification (right) of histological cross sections derived from TA muscles and TA muscle weight described in C. Scale bars = 100 μm. (E) Zbed6, Dkk3, Fbxo32 and Murf1 mRNA expression levels described in C. n = 3. (F) Representative western blotting and quantification of Dkk3, Fbxo32, Murf1, phosphorylated protein levels of FoxO3 and total FoxO3 in muscle described in C. n = 3. GAPDH served as internal control. Data are expressed as mean ± SEM; * p < 0.05, ** p < 0.01.

Article Snippet: The C2C12 cell line (ATCC) was cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 4.5 g/L glucose (11955065, Gibco, United States), 10% foetal bovine serum (FBS) (10099141C, Gibco, United States) and 1% penicillin–streptomycin solution (P1400, Solarbio, Beijing, China).

Techniques: Over Expression, In Vitro, Control, Plasmid Preparation, Infection, Negative Control, Staining, Expressing, Virus, Injection, Derivative Assay, Muscles, Western Blot

Journal: Molecular Medicine Reports

Article Title: Glucose, glutamine, lactic acid and α-ketoglutarate restore metabolic disturbances and atrophic changes in energy-deprived muscle cells

doi: 10.3892/mmr.2025.13562

Figure Lengend Snippet: Effect of single nutrient supplementation on metabolic activity. (A) Cells were incubated in regular DMEM supplemented with the indicated nutrient for 24 h. (B and C) C2C12 myotubes were cultured in starvation medium only or with the indicated nutrient for (B) 5 or (C) 24 h. Albumin and NaOH were added as vehicle controls in both regular and starvation media in the experiment for the fatty acid (PA and OA). Metabolic activity was assessed using the MTT assay. Values are expressed as fold-change compared with the values of the control cells incubated in regular DMEM. Values are presented as the mean ± SEM (n=3-4). ***P<0.001 vs. vehicle control; § P<0.05, §§ P<0.01, §§§ P<0.001 vs. starved cells in the same group. Cont, control; Vehicle, vehicle control; Stv, starvation; Glc, glucose; Gln, glutamine; Glu, glutamic acid; Leu, leucine; Val, valine; LA, lactate; βOHB, β-hydroxy butyric acid; αKG, α-ketoglutarate; PA, palmitic acid; OA, oleic acid.

Article Snippet: Mouse C2C12 myoblast cell line (RCB0987) was purchased from the RIKEN BioResource Research Center (Tsukuba, Japan).

Techniques: Activity Assay, Incubation, Cell Culture, MTT Assay, Control

Journal: Molecular Medicine Reports

Article Title: Glucose, glutamine, lactic acid and α-ketoglutarate restore metabolic disturbances and atrophic changes in energy-deprived muscle cells

doi: 10.3892/mmr.2025.13562

Figure Lengend Snippet: Effect of single nutrient supplementation on ATP production. C2C12 myotubes were incubated in starvation medium with or without the indicated nutrients for 24 h. The ATP production rates from glycolysis (Glyco-ATP) and OxPhos (OxPhos-ATP) were determined. Values are expressed as fold-change compared with the values of the control cells incubated in regular DMEM. Values are presented as mean ± SEM (n=3-4). **P<0.01, ***P<0.001 vs. control cells for total ATP production; §§§ P<0.001 vs. starved cells for Glyco-ATP production. Con, control; Glyco-ATP, glycolytic ATP; OxPhos, oxidative phosphorylation; Stv, starvation.

Article Snippet: Mouse C2C12 myoblast cell line (RCB0987) was purchased from the RIKEN BioResource Research Center (Tsukuba, Japan).

Techniques: Incubation, Control, Phospho-proteomics

Journal: Molecular Medicine Reports

Article Title: Glucose, glutamine, lactic acid and α-ketoglutarate restore metabolic disturbances and atrophic changes in energy-deprived muscle cells

doi: 10.3892/mmr.2025.13562

Figure Lengend Snippet: Effect of single nutrient supplementation on metabolic activity. (A) Cells were incubated in regular DMEM supplemented with the indicated nutrient for 24 h. (B and C) C2C12 myotubes were cultured in starvation medium only or with the indicated nutrient for (B) 5 or (C) 24 h. Albumin and NaOH were added as vehicle controls in both regular and starvation media in the experiment for the fatty acid (PA and OA). Metabolic activity was assessed using the MTT assay. Values are expressed as fold-change compared with the values of the control cells incubated in regular DMEM. Values are presented as the mean ± SEM (n=3-4). ***P<0.001 vs. vehicle control; § P<0.05, §§ P<0.01, §§§ P<0.001 vs. starved cells in the same group. Cont, control; Vehicle, vehicle control; Stv, starvation; Glc, glucose; Gln, glutamine; Glu, glutamic acid; Leu, leucine; Val, valine; LA, lactate; βOHB, β-hydroxy butyric acid; αKG, α-ketoglutarate; PA, palmitic acid; OA, oleic acid.

Article Snippet: To induce differentiation of myoblasts into myotubes, C2C12 cells at 80% confluency were maintained in the differentiation medium (DMEM containing 2% horse serum; Thermo Fisher Scientific, Tokyo, Japan) for 6 days.

Techniques: Activity Assay, Incubation, Cell Culture, MTT Assay, Control

Journal: Molecular Medicine Reports

Article Title: Glucose, glutamine, lactic acid and α-ketoglutarate restore metabolic disturbances and atrophic changes in energy-deprived muscle cells

doi: 10.3892/mmr.2025.13562

Figure Lengend Snippet: Effect of single nutrient supplementation on ATP production. C2C12 myotubes were incubated in starvation medium with or without the indicated nutrients for 24 h. The ATP production rates from glycolysis (Glyco-ATP) and OxPhos (OxPhos-ATP) were determined. Values are expressed as fold-change compared with the values of the control cells incubated in regular DMEM. Values are presented as mean ± SEM (n=3-4). **P<0.01, ***P<0.001 vs. control cells for total ATP production; §§§ P<0.001 vs. starved cells for Glyco-ATP production. Con, control; Glyco-ATP, glycolytic ATP; OxPhos, oxidative phosphorylation; Stv, starvation.

Article Snippet: To induce differentiation of myoblasts into myotubes, C2C12 cells at 80% confluency were maintained in the differentiation medium (DMEM containing 2% horse serum; Thermo Fisher Scientific, Tokyo, Japan) for 6 days.

Techniques: Incubation, Control, Phospho-proteomics

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: ZBED6 Knockout Prevents Ageing‐ and Dexamethasone‐Induced Muscle Atrophy via Dkk3 in Pig and Mice

doi: 10.1002/jcsm.13829

Figure Lengend Snippet: Zbed6 controls Dkk3 transcription in ageing‐induced muscle atrophy. (A) Representative H&E staining (top), Sirius (middle), fibre‐type staining (bottom) and quantification (right) of histological cross sections in GM tissues of Zbed6 −/− and controls of 18 months old mice. Myosin heavy chain type I and IIa (red), IIb (green) and DAPI (blue). Representative images are shown. Scale bar = 200 μm. n = 3; (B) Dkk3, Fbxo32 and Murf1 mRNA expression levels in mice described in A. n = 3. (C) Representative western blotting and quantification of Dkk3, Fbxo32 and Murf1 in mice described in A. GAPDH served as internal control. n = 3. (D) Representative western blotting of Zbed6 in WT and Zbed6‐KO C2C12 cells ( n = 3) and representative images for fibre diameter and area of myotubes (myogenic differentiation takes 72 h). Red indicated myosin heavy chain (MyHC) immunofluorescent staining and DAPI (blue). Scale bars = 100 μm. (E) Dkk3, Fbxo32 and Murf1 mRNA expression levels decreased after Zbed6 depleted. n = 4. GAPDH served as internal control. Data are expressed as mean ± SEM; * p < 0.05, ** p < 0.01.

Article Snippet: To induce atrophy, the myotubes that had undergone differentiation for 72 h were incubated with 100 μM Dex (HY‐14648, MCE, United States) for 24 h. The coding sequence of Zbed6 in C2C12 cells was targeted using CRISPR/Cas9 technology.

Techniques: Staining, Expressing, Western Blot, Control

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: ZBED6 Knockout Prevents Ageing‐ and Dexamethasone‐Induced Muscle Atrophy via Dkk3 in Pig and Mice

doi: 10.1002/jcsm.13829

Figure Lengend Snippet: Overexpression of Dkk3 downregulation Zbed6 deficiency‐mediated against muscle atrophy in vivo and vitro. (A) Schematic representation of mouse model. AAV9‐myo2A‐GFP and AAV‐myo2A‐Dkk3 were injected in TA muscle of the left and right legs of the same mice (3‐month‐old) in Zbed6 f/f and Zbed6 −/− mice for one month, and the following experimental setup ( n = 3 mice of Zbed6 f/f ; n = 4 mice of Zbed6 −/− ) (left). Representative western blotting and quantification of Flag‐Dkk3 (right). (B) Dkk3, Fbxo32 and Murf1 mRNA expression levels in mice described in A. (C) Representative H&E staining (left), average CSA (upper right) and TA muscle weight in mice described in A. Scale bars = 200 μm. (D) Schematic representation of cultured myotubes using 2% horse serum; the differentiation was induced to form myotubes for 72 h and then infected by adenovirus‐encoding Dkk3 or control vector for 48 h. (E) Representative images and quantification (left) for fibre diameter and area of myotubes infected for 120 hours in C2C12 described in D. Red indicated myosin heavy chain (MyHC) immunofluorescent staining and DAPI (blue). Scale bars = 100 μm. Dkk3, Murf1 and Fbxo32 mRNA expression level (right). n = 3. (F) Representative western blotting and quantification of Zbed6, Dkk3, Fbxo32, Murf1, phosphorylated protein levels of FoxO3 and total FoxO3 in C2C12 described in D. GAPDH served as internal control. Data are expressed as mean ± SEM; * p < 0.05, ** p < 0.01.

Article Snippet: To induce atrophy, the myotubes that had undergone differentiation for 72 h were incubated with 100 μM Dex (HY‐14648, MCE, United States) for 24 h. The coding sequence of Zbed6 in C2C12 cells was targeted using CRISPR/Cas9 technology.

Techniques: Over Expression, In Vivo, Injection, Western Blot, Expressing, Staining, Cell Culture, Infection, Control, Plasmid Preparation

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: ZBED6 Knockout Prevents Ageing‐ and Dexamethasone‐Induced Muscle Atrophy via Dkk3 in Pig and Mice

doi: 10.1002/jcsm.13829

Figure Lengend Snippet: Zbed6 depletion protects dexamethasone‐induced myotubes and muscles atrophy. (A) Schematic representation of Dex injection, representative H&E staining and quantification of histological cross sections derived from Zbed6 −/− and controls (3‐month‐old) TA muscles, and mice was injected with or without 15 mg/kg/day of Dex for 10 days. Scale bars = 100 μm. n = 6. (B) mRNA expression levels of Zbed6, Dkk3, Murf1 and Fbxo32 described in A. n = 6. (C) Representative western blotting and quantification of Zbed6, Dkk3, Murf1 and Fbxo32 in mice described in A. n = 3. (D) Representative images (top) and quantification (bottom) for fibre diameter and area of myotubes. WT and Zbed6‐KO C2C12 myotubes with or without Dex; the myotubes were treated with 100 μM Dex for 24 h. Red indicated myosin heavy chain (MyHC) immunofluorescent staining and DAPI (blue). Scale bars = 100 μm. n = 3. (E, F) mRNA and protein expression levels of Zbed6, Dkk3, Murf1 and Fbxo32 described in D. n = 3. GAPDH served as internal control. Data are expressed as mean ± SEM; * p < 0.05, ** p < 0.01.

Article Snippet: To induce atrophy, the myotubes that had undergone differentiation for 72 h were incubated with 100 μM Dex (HY‐14648, MCE, United States) for 24 h. The coding sequence of Zbed6 in C2C12 cells was targeted using CRISPR/Cas9 technology.

Techniques: Muscles, Injection, Staining, Derivative Assay, Expressing, Western Blot, Control

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: ZBED6 Knockout Prevents Ageing‐ and Dexamethasone‐Induced Muscle Atrophy via Dkk3 in Pig and Mice

doi: 10.1002/jcsm.13829

Figure Lengend Snippet: Overexpression of Zbed6 accelerates muscle atrophy in vitro and vivo . (A) Representative images (left) and quantification (right) of myotubes. Primary myotubes overexpressing Zbed6 by adenovirus‐encoding Zbed6 or control vector, 24 h after infection, the myotubes infection with Si‐Dkk3 or negative‐control (NC). Red indicated myosin heavy chain (MyhC) immunofluorescent staining and DAPI (blue). Scale bars = 200 μm. n = 3. (B) Zbed6, Dkk3, Murf1 and Fbxo32 mRNA expression level in C2C12 described in A. n = 4. (C) Schematic representation of virus and Dex injection. AAV9‐myo2A‐GFP and AAV‐myo2A‐Zbed6 were injected in TA muscle of the left and right legs of the same mice (3‐month‐old). One month later, the mice were intraperitoneally injected with or without 15 mg/kg/day of dexamethasone for 10 days. n = 3. (D) Representative H&E staining (left) and quantification (right) of histological cross sections derived from TA muscles and TA muscle weight described in C. Scale bars = 100 μm. (E) Zbed6, Dkk3, Fbxo32 and Murf1 mRNA expression levels described in C. n = 3. (F) Representative western blotting and quantification of Dkk3, Fbxo32, Murf1, phosphorylated protein levels of FoxO3 and total FoxO3 in muscle described in C. n = 3. GAPDH served as internal control. Data are expressed as mean ± SEM; * p < 0.05, ** p < 0.01.

Article Snippet: To induce atrophy, the myotubes that had undergone differentiation for 72 h were incubated with 100 μM Dex (HY‐14648, MCE, United States) for 24 h. The coding sequence of Zbed6 in C2C12 cells was targeted using CRISPR/Cas9 technology.

Techniques: Over Expression, In Vitro, Control, Plasmid Preparation, Infection, Negative Control, Staining, Expressing, Virus, Injection, Derivative Assay, Muscles, Western Blot