mouse skeletal muscle derived myoblasts  (ATCC)

Journal: Molecular Medicine Reports

Article Title: 20(s)-ginseonside-Rg3 modulation of AMPK/FoxO3 signaling to attenuate mitochondrial dysfunction in a dexamethasone-injured C2C12 myotube-based model of skeletal atrophy in vitro

doi: 10.3892/mmr.2021.11945

Figure Lengend Snippet: Muscle atrophy and mitochondrial dysfunction induced by DEX. (A) After 24 or 48-h treatment with various doses of DEX (25, 50, 100 and 200 µM), no effect on C2C12 myotube viability was observed at DEX concentrations <200 µM. **P<0.01 vs. CON (24 h); ## P<0.01 vs. CON (48 h). Results were analyzed via one-way ANOVA with data expressed as the mean ± standard deviation (n=3). (B) DEX-treated C2C12 myotube atrogen-1 and MuRF1 mRNA levels were higher compared with corresponding levels in control cells. **P<0.01 vs. CON. (MuRF1); ## P<0.01 vs. CON (atrogin-1). Results were analyzed via Student's t-test, with data expressed as the mean ± standard deviation (n=3). (C) Intracellular ATP level was reduced following 24-h treatment with various DEX doses (25, 50, 100 and 200 µM). *P<0.05 and **P<0.01 vs. CON. Results were analyzed via one-way ANOVA, with data expressed as the mean ± standard deviation (n=3). (D) Basal respiration (OCR) measured by MitoXpress ® Xtra Oxygen Consumption Assay was reduced at 24 h following administration of various doses of DEX (25, 50, 100 and 200 µM). *P<0.05 and **P<0.01 vs. CON. Results were analyzed via one-way ANOVA, with data expressed as the mean ± standard deviation (n=3). (E) Reduction of intracellular ATP level following DEX incubation for various time points in hours. *P<0.05 vs. CON. Results were analyzed via one-way ANOVA, with data expressed as the mean ± standard deviation (n=3). (F) Reduction of OCR following DEX administration for various time points in hours. *P<0.05 vs. CON. Results were analyzed via one-way ANOVA, with data expressed as the mean ± standard deviation (n=3). DEX, dexamethasone; CON, 0 µM DEX; MuRF1, muscle RING finger-1; atrogin-1, muscle atrophy F-box protein; OCR, oxygen consumption rate.

Article Snippet: Chemicals, reagents and kits were obtained from commercial sources as follows: S-Rg3 (Urchem Sinopharm Chemical Reagent Co., Ltd.), dexamethasone (DEX)and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich; Merck KGaA), mouse skeletal muscle-derived C2C12 myoblasts (American Type Culture Collection), fetal bovine serum (FBS; Clark BioScience), horse serum (Gibco; Thermo Fisher Scientific, Inc.), antibodies against cleaved caspase-3 (cat. no. 9661), phosphorylated-AMPK (p-AMPK; cat. no. 2535), AMPK (cat. no. 2532), Bcl-2 (cat. no. 3498), Bax (cat. no. 2772), FoxO3 (cat. no. 2497), histone H3 (cat. no. 4499) and β-tubulin (cat. no. 2146) were obtained from Cell Signaling Technology, Inc.; atrogin-1 antibody (cat. no. ab168372) and total OXPHOS Rodent WB Antibody Cocktail (anti-complex I, II, III, IV, V; cat. no. ab110413) were obtained from Abcam; antibody specific for muscle-specific RING finger 1 (MuRF1; cat. no. bs-2539R) was purchased from BIOSS.

Techniques: Standard Deviation, Control, Incubation

Journal: Molecular Medicine Reports

Article Title: 20(s)-ginseonside-Rg3 modulation of AMPK/FoxO3 signaling to attenuate mitochondrial dysfunction in a dexamethasone-injured C2C12 myotube-based model of skeletal atrophy in vitro

doi: 10.3892/mmr.2021.11945

Figure Lengend Snippet: S-Rg3 reversal of DEX-induced C2C12 myotube atrophy. (A) After 24-h treatment of C2C12 myotubes with S-Rg3 (0.02, 0.2 and 2 µM) and DEX, MTT assays were conducted to measure viability of C2C12 myotube cells. **P<0.01 vs. CON; # P<0.01 and ## P<0.01 vs. DEX. Results were analyzed via one-way ANOVA, with data expressed as the mean ± standard deviation (n=3). (B) To further analyze S-Rg3 treatment effects on skeletal muscle cell atrophy, atrogin-1 and MuRF1 expression levels were assessed via western blot analysis. (C and D) Relative atrogin-1 and MuRF1 expression levels were quantified via densitometric analysis, with β-tubulin serving as loading control. *P<0.05 vs. CON; # P<0.05 and ## P<0.01 vs. DEX. Results were analyzed via one-way ANOVA, with data expressed as the mean ± standard deviation (n=3). (E) Following treatment of C2C12 myotubes with S-Rg3 and/or DEX for 24 h, cleaved caspase-3, Bcl-2 and Bax levels were measured via western blot analysis. (F) Relative expression levels of cleaved caspase-3 and Bcl-2/Bax were semi-quantified by densitometric analyses based on β-tubulin as loading control. Results were analyzed using a one-way ANOVA. Data are shown as the mean ± standard deviation (n=3). **P<0.01, ***P<0.001 vs. CON; # P<0.05, ## P<0.01 vs. DEX. S-Rg3, 20(S)-ginsenoside Rg3; DEX, dexamethasone; MuRF1, muscle RING finger-1; atrogin-1, muscle atrophy F-box protein; CON, control group.

Article Snippet: Chemicals, reagents and kits were obtained from commercial sources as follows: S-Rg3 (Urchem Sinopharm Chemical Reagent Co., Ltd.), dexamethasone (DEX)and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich; Merck KGaA), mouse skeletal muscle-derived C2C12 myoblasts (American Type Culture Collection), fetal bovine serum (FBS; Clark BioScience), horse serum (Gibco; Thermo Fisher Scientific, Inc.), antibodies against cleaved caspase-3 (cat. no. 9661), phosphorylated-AMPK (p-AMPK; cat. no. 2535), AMPK (cat. no. 2532), Bcl-2 (cat. no. 3498), Bax (cat. no. 2772), FoxO3 (cat. no. 2497), histone H3 (cat. no. 4499) and β-tubulin (cat. no. 2146) were obtained from Cell Signaling Technology, Inc.; atrogin-1 antibody (cat. no. ab168372) and total OXPHOS Rodent WB Antibody Cocktail (anti-complex I, II, III, IV, V; cat. no. ab110413) were obtained from Abcam; antibody specific for muscle-specific RING finger 1 (MuRF1; cat. no. bs-2539R) was purchased from BIOSS.

Techniques: Standard Deviation, Expressing, Western Blot, Control

Journal: Molecular Medicine Reports

Article Title: 20(s)-ginseonside-Rg3 modulation of AMPK/FoxO3 signaling to attenuate mitochondrial dysfunction in a dexamethasone-injured C2C12 myotube-based model of skeletal atrophy in vitro

doi: 10.3892/mmr.2021.11945

Figure Lengend Snippet: S-Rg3 reversal of mitochondrial dysfunction induced by DEX. (A) DEX-injured C2C12 myotube intracellular ATP level was increased by S-Rg3 treatment. *P<0.05 vs. CON; # P<0.05 and ## P<0.01 vs. DEX. Results were analyzed via one-way ANOVA, with data expressed as the mean ± standard deviation (n=3). (B) OCR as measured by MitoXpress ® Xtra Oxygen Consumption Assay measurement showing OCR decrease following 6-h DEX administration that was restored following administration of S-Rg3. *P<0.05 vs. CON; # P<0.05 and ## P<0.01 vs. DEX. Results were analyzed via one-way ANOVA, with data expressed as the mean ± standard deviation (n=3). (C) After 6-h DEX and/or S-Rg3 treatments, mitochondrial membrane potential was assessed following incubation of C2C12 myotubes with the JC-1 probe followed by flow cytometric analysis. (D) Bar graph representing cells staining positive for JC-1 monomer then analyzed as in (C). ***P<0.001 vs. CON; ## P<0.01 and ### P<0.001 vs. DEX. Results were analyzed via one-way ANOVA, with data expressed as the mean ± standard deviation (n=3). (E) Representative western blots of DEX-injured C2C12 myotube complex I, II, III, IV and V proteins. (F) Relative expression of complex I, II, III, IV and V proteins quantified by densitometric analysis, with β-tubulin serving as loading control. Data are expressed as the mean ± standard deviation (n=3). **P<0.01 vs. CON; # P<0.05 and ## P<0.01 vs. DEX. Results were analyzed via one-way ANOVA, with data expressed as the mean ± standard deviation (n=3). S-Rg3, 20(S)-ginsenoside Rg3; DEX, dexamethasone; OCR, oxygen consumption rate; CON, control group.

Article Snippet: Chemicals, reagents and kits were obtained from commercial sources as follows: S-Rg3 (Urchem Sinopharm Chemical Reagent Co., Ltd.), dexamethasone (DEX)and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich; Merck KGaA), mouse skeletal muscle-derived C2C12 myoblasts (American Type Culture Collection), fetal bovine serum (FBS; Clark BioScience), horse serum (Gibco; Thermo Fisher Scientific, Inc.), antibodies against cleaved caspase-3 (cat. no. 9661), phosphorylated-AMPK (p-AMPK; cat. no. 2535), AMPK (cat. no. 2532), Bcl-2 (cat. no. 3498), Bax (cat. no. 2772), FoxO3 (cat. no. 2497), histone H3 (cat. no. 4499) and β-tubulin (cat. no. 2146) were obtained from Cell Signaling Technology, Inc.; atrogin-1 antibody (cat. no. ab168372) and total OXPHOS Rodent WB Antibody Cocktail (anti-complex I, II, III, IV, V; cat. no. ab110413) were obtained from Abcam; antibody specific for muscle-specific RING finger 1 (MuRF1; cat. no. bs-2539R) was purchased from BIOSS.

Techniques: Standard Deviation, Membrane, Incubation, Staining, Western Blot, Expressing, Control

Journal: Molecular Medicine Reports

Article Title: 20(s)-ginseonside-Rg3 modulation of AMPK/FoxO3 signaling to attenuate mitochondrial dysfunction in a dexamethasone-injured C2C12 myotube-based model of skeletal atrophy in vitro

doi: 10.3892/mmr.2021.11945

Figure Lengend Snippet: S-Rg3 inhibition of the AMPK/FOXO3 signaling pathway induced in C2C12 myotubes by DEX. (A) After 6-h S-Rg3 treatment, p-AMPK and AMPK levels in DEX-injured C2C12 myotubes were detected by western blot analysis, with β-tubulin serving as loading control. (B) Relative expression of p-AMPK/AMPK quantified using densitometric analysis, *P<0.05 vs. CON; # P<0.05 and ## P<0.01 vs. DEX. Results were analyzed via one-way ANOVA, with data expressed as the mean ± standard deviation (n=3). (C) To further explore S-Rg3 effects, nuclear FoxO3 expression was studied via western blot analysis, with histone H3 serving as the nuclear protein loading control. Relative FoxO3 expression levels were semi-quantified via densitometric analysis. **P<0.01 vs. CON; # P<0.05, ## P<0.01 vs. DEX. Results were analyzed via one-way ANOVA, with data expressed as the mean ± standard deviation (n=3). (D) To further explore S-Rg3 effects, cytoplasmic FoxO3 expression was studied via western blot analysis, with β-tubulin serving as the cytoplasmic protein loading control. Relative FoxO3 expression levels were semi-quantified via densitometric analysis. *P<0.05 vs. CON; # P<0.05, ## P<0.01 vs. DEX. Results were analyzed via one-way ANOVA, with data expressed as the mean ± standard deviation (n=3). S-Rg3, 20(S)-ginsenoside Rg3; AMPK, AMP-activated protein kinase; FoxO3, forkhead box O3; p-, phosphorylated; DEX, dexamethasone; CON, control group.

Article Snippet: Chemicals, reagents and kits were obtained from commercial sources as follows: S-Rg3 (Urchem Sinopharm Chemical Reagent Co., Ltd.), dexamethasone (DEX)and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich; Merck KGaA), mouse skeletal muscle-derived C2C12 myoblasts (American Type Culture Collection), fetal bovine serum (FBS; Clark BioScience), horse serum (Gibco; Thermo Fisher Scientific, Inc.), antibodies against cleaved caspase-3 (cat. no. 9661), phosphorylated-AMPK (p-AMPK; cat. no. 2535), AMPK (cat. no. 2532), Bcl-2 (cat. no. 3498), Bax (cat. no. 2772), FoxO3 (cat. no. 2497), histone H3 (cat. no. 4499) and β-tubulin (cat. no. 2146) were obtained from Cell Signaling Technology, Inc.; atrogin-1 antibody (cat. no. ab168372) and total OXPHOS Rodent WB Antibody Cocktail (anti-complex I, II, III, IV, V; cat. no. ab110413) were obtained from Abcam; antibody specific for muscle-specific RING finger 1 (MuRF1; cat. no. bs-2539R) was purchased from BIOSS.

Techniques: Inhibition, Western Blot, Control, Expressing, Standard Deviation