c2c12 myoblasts  (ATCC)

Journal: The FASEB Journal

Article Title: NOR ‐1 Overexpression Elevates Myoglobin Expression via PERM1 and Enhances Mitochondrial Function and Endurance in Skeletal Muscles of Aged Mice

doi: 10.1096/fj.202500375R

Figure Lengend Snippet: NOR‐1 overexpression elevates metabolic gene expression in C2C12 myotubes. (A) Vector map of AAV9 used for NOR‐1 overexpression. (B) Images showing GFP+ myotubes after AAV9 transduction. The scale bar is 200 μm. (C) NOR‐1 gene expression 7 days after AAV9 transduction ( n = 6). (D) Metabolic gene expression in C2C12 myotubes after NOR‐1 overexpression ( n = 6). (E) Experimental design: C2C12 myotubes were differentiated for 3 days before being transduced with AAVs to express either NOR‐1 or GFP control protein. 5 days after transduction, NOR‐1‐GFP and control‐GFP expressing myotubes were treated with either a PERM1 targeting or negative control siRNA for 2 days before being harvested. Expression of (F) PERM1, (G) CKMT2, and (H) myoglobin after AAV transduction and siRNA transfection ( n = 6). HPRT was used as a housekeeping gene. Data are presented as Mean ± SEM. (C and D) are analyzed using unpaired t ‐test or unpaired t ‐test with Welch's correction when unequal variance was detected. (F–H) were analyzed using One‐Way ANOVA or Brown‐Forsythe and Welch ANOVA when unequal variance was detected. Statistical significance was set to p < 0.05. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Different letters (A, B, C) denote significant differences between groups. (A) The vector map was created with Vectorbuilder. (E) Made with Biorender.com (Created in BioRender. Paez, H. (2024) https://BioRender.com/g53o551 ).

Article Snippet: To induce differentiation, C2C12 myoblasts were grown to 70% confluence and GM was replaced with differentiation media (DM) containing DMEM supplemented with 2% heat‐inactivated horse serum (Gibco) and 1% antibiotic/antimycotic.

Techniques: Over Expression, Gene Expression, Plasmid Preparation, Transduction, Control, Expressing, Negative Control, Transfection

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Transcriptional Intermediary Factor 1γ–Induced Irisin in Skeletal Muscle Attenuates Renal Fibrosis in Diabetic Nephropathy

doi: 10.1002/jcsm.13810

Figure Lengend Snippet: Effect of TIF1γ on irisin expression and secretion in C2C12 cells. (A & B) Experimental in vitro scheme for C2C12 differentiation. (C & D) Changes in irisin expression and secretion following TIF1γ plasmid (2 μg) transfection during C2C12 differentiation. Statistical significance was determined using a one‐way analysis of variance followed by Tukey's multiple comparison test. Data are presented as mean ± SEM. * p < 0.05. TIF1γ, transcriptional intermediary factor 1γ; C2C12, murine myoblast cells; SEM, standard error of the mean.

Article Snippet: The C2C12 mouse skeletal muscle myoblasts were purchased from the American Type Culture (ATCC, USA).

Techniques: Expressing, In Vitro, Plasmid Preparation, Transfection, Comparison

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Transcriptional Intermediary Factor 1γ–Induced Irisin in Skeletal Muscle Attenuates Renal Fibrosis in Diabetic Nephropathy

doi: 10.1002/jcsm.13810

Figure Lengend Snippet: Effect of TIF1γ on palmitate treatment in C2C12 cells and the paracrine effect of TIF1γ‐induced irisin on palmitate‐treated HK‐2 cells. (A) Changes in irisin, TIF1γ, PGC‐1α and pAkt expression following palmitate (100 μM) treatment in the presence or absence of TIF1γ (2 μg). (B) Changes in irisin secretion following palmitate (100 μM) treatment with or without TIF1γ (2 μg). (C) Changes in EMT markers in palmitate (100 μM)‐treated HK‐2 cells induced by CM from TIF1γ‐transfected C2C12 cells. (D) Western blot analysis of FNDC5 protein levels in C2C12 cells transfected with control or FNDC5‐specific siRNAs (#1–5) for 48 h. (E) Irisin levels in CM from C2C12 cells transfected with 25 nmol siRNA‐FNDC5 and 2 μg TIF1γ, measured by ELISA. (F) Changes in EMT markers in palmitate (100 μM)‐treated HK‐2 cells induced by CM from C2C12 cells transfected with 25 nmol siRNA‐FNDC5 and TIF1γ. Each protein expression level was normalized to α‐tubulin or β‐actin (A, C, D, F). TIF1γ, transcriptional intermediary factor 1γ; HK‐2, human kidney 2 cells; C2C12, murine myoblast cells; PGC‐1α, peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha; pAkt, phosphorylated protein kinase B; EMT, epithelial–mesenchymal transition; BMP‐7, bone morphogenetic protein 7; α‐SMA, alpha‐smooth muscle actin; pSmad2/3, phosphorylated Smad2/3; CM, conditioned medium; siRNA, signal interfering RNA.

Article Snippet: The C2C12 mouse skeletal muscle myoblasts were purchased from the American Type Culture (ATCC, USA).

Techniques: Expressing, Transfection, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: HD6277 Suppresses Muscle Atrophy by Promoting Myogenic Factors and Inhibiting Proteolysis in Aged Mice

doi: 10.1002/jcsm.13805

Figure Lengend Snippet: HD6277 accelerated myotube formation in an Akt‐dependent manner in C2C12 myoblasts. (A,B) The cells were differentiated with various doses of HD6277 (0, 10 or 25 μM) or HD6277 plus GW1100 (10 μM) for 2 days, and then, the phosphorylated Akt level was determined by Western blotting. (C) The cells were differentiated with HD6277 (25 μM) with or without an Akt inhibitor (1 or 2 μM) for 2 days, and then, the transcript level of Mef2c was analysed by qPCR. (D) MyoG and MyHC levels were determined by Western blotting. (E) The MyHC‐positive area and myotube width were calculated in MyHC‐stained cells (green fluorescence). Scale bar: 275 μm. Akti, Akt inhibitor; GW, GW1100; HD, HD6277; Veh, vehicle. Error bars indicate the mean ± SD (* p < 0.05; ANOVA with post hoc t test).

Article Snippet: Total RNA was isolated from C2C12 myoblasts and muscle tissues using Qiazol Lysis Reagent (Qiagen, Hilden, Germany) and used to synthesize complementary deoxyribonucleic acid (cDNA).

Techniques: Western Blot, Staining, Fluorescence

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: HD6277 Suppresses Muscle Atrophy by Promoting Myogenic Factors and Inhibiting Proteolysis in Aged Mice

doi: 10.1002/jcsm.13805

Figure Lengend Snippet: HD6277 inhibited the palmitate‐mediated reduction in myotube formation in an Akt‐dependent manner in C2C12 myoblasts. (A,B) The cells were stimulated with palmitate (200 μM), palmitate plus HD6277 (10 or 25 μM) or palmitate plus HD6277 with GW1100 (10 μM). The phosphorylated Akt level was analysed by Western blotting. (C) The cells were stimulated with palmitate (200 μM), palmitate plus HD6277 (25 μM) or palmitate plus HD6277 with Akt inhibitor (2 μM) and then differentiated for 2 days. The transcript levels of Mef2c and Myf5 were analysed by qPCR. (D) MyoG and MyHC levels were analysed by Western blotting. (E) MyHC‐stained cells (green fluorescence) were observed under a fluorescence microscope to calculate the MyHC‐positive area and myotube width. Scale bar: 275 μm. Akti, Akt inhibitor; GW, GW1100; HD, HD6277; PA, palmitate; Veh, vehicle. Error bars indicate the mean ± SD (* p < 0.05; ANOVA with post hoc t test).

Article Snippet: Total RNA was isolated from C2C12 myoblasts and muscle tissues using Qiazol Lysis Reagent (Qiagen, Hilden, Germany) and used to synthesize complementary deoxyribonucleic acid (cDNA).

Techniques: Western Blot, Staining, Fluorescence, Microscopy

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: HD6277 Suppresses Muscle Atrophy by Promoting Myogenic Factors and Inhibiting Proteolysis in Aged Mice

doi: 10.1002/jcsm.13805

Figure Lengend Snippet: HD6277 suppressed palmitate‐mediated cytotoxicity in an Akt‐dependent manner in C2C12 myoblasts. (A,D) The cells were stimulated with palmitate (200 μM), palmitate plus HD6277 (10 or 25 μM) or palmitate plus HD6277 with Akt inhibitor (1 or 2 μM), after which the Bax/Bcl2 ratio and cleaved caspase 3 level were determined by Western blotting. (B,E) Cell viability was measured using the EZ‐Cytox solution. (C,F) DNA fragmentation was visualized using a TUNEL assay kit. Scale bar: 125 μm. TUNEL‐positive cells (red fluorescence) were observed and counted under a fluorescence microscope. Akti, Akt inhibitor; C.Cas‐3, cleaved caspase 3; HD, HD6277; PA, palmitate; Veh, vehicle. Error bars indicate the mean ± SD (* p < 0.05; ANOVA with post hoc t test).

Article Snippet: Total RNA was isolated from C2C12 myoblasts and muscle tissues using Qiazol Lysis Reagent (Qiagen, Hilden, Germany) and used to synthesize complementary deoxyribonucleic acid (cDNA).

Techniques: Western Blot, TUNEL Assay, Fluorescence, Microscopy

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: HD6277 Suppresses Muscle Atrophy by Promoting Myogenic Factors and Inhibiting Proteolysis in Aged Mice

doi: 10.1002/jcsm.13805

Figure Lengend Snippet: HD6277 reduced the palmitate‐induced proteolytic processes in an Akt‐dependent manner in C2C12 myotubes. (A,B) Fully differentiated cells were stimulated with palmitate (400 μM), palmitate plus HD6277 (10 or 25 μM) or palmitate plus HD6277 with GW1100 (10 μM), and then, the phosphorylated Akt level was determined by Western blotting. (C–F) Fully differentiated cells were stimulated with palmitate (400 μM), palmitate plus HD6277 (10 or 25 μM) or palmitate plus HD6277 with Akt inhibitor (2 μM). Western blotting was then performed to analyse the levels of phosphorylated FOXO1A, atrogin‐1, MuRF1 and ubiquitination. Akti, Akt inhibitor; GW, GW1100; HD, HD6277; PA, palmitate; Veh, vehicle. Error bars indicate the mean ± SD (* p < 0.05; ANOVA with post hoc t test).

Article Snippet: Total RNA was isolated from C2C12 myoblasts and muscle tissues using Qiazol Lysis Reagent (Qiagen, Hilden, Germany) and used to synthesize complementary deoxyribonucleic acid (cDNA).

Techniques: Western Blot

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: HD6277 Suppresses Muscle Atrophy by Promoting Myogenic Factors and Inhibiting Proteolysis in Aged Mice

doi: 10.1002/jcsm.13805

Figure Lengend Snippet: HD6277 inhibited palmitate‐induced cytotoxicity in an Akt‐dependent manner in C2C12 myotubes. (A,D) Fully differentiated cells were incubated with palmitate (400 μM), palmitate plus HD6277 (10 or 25 μM) or palmitate plus HD6277 with Akt inhibitor (2 μM), and then, Western blotting was performed to determine the levels of the Bax/Bcl2 ratio and cleaved caspase 3. (B,E) Cell viability was measured using EZ‐Cytox solution. (C,F) DNA fragmentation was analysed by the TUNEL assay. Scale bar: 125 μm. TUNEL‐positive cells (red fluorescence) were calculated under a fluorescence microscope. Akti, Akt inhibitor; C.Cas‐3, cleaved caspase 3; HD, HD6277; PA, palmitate; Veh, vehicle. Error bars indicate the mean ± SD (* p < 0.05; ANOVA with post hoc t test).

Article Snippet: Total RNA was isolated from C2C12 myoblasts and muscle tissues using Qiazol Lysis Reagent (Qiagen, Hilden, Germany) and used to synthesize complementary deoxyribonucleic acid (cDNA).

Techniques: Incubation, Western Blot, TUNEL Assay, Fluorescence, Microscopy

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: HD6277 Suppresses Muscle Atrophy by Promoting Myogenic Factors and Inhibiting Proteolysis in Aged Mice

doi: 10.1002/jcsm.13805

Figure Lengend Snippet: Schematic diagram of HD6277 functions in muscles. The HD6277‐Akt axis enhanced myogenic factors, reduced proteolytic processes and inhibited cytotoxicity in C2C12 cells and muscle tissues from obese and aged mice. Furthermore, HD6277 administration improved muscle strength and the CSA of muscle fibres in aged mice.

Article Snippet: Total RNA was isolated from C2C12 myoblasts and muscle tissues using Qiazol Lysis Reagent (Qiagen, Hilden, Germany) and used to synthesize complementary deoxyribonucleic acid (cDNA).

Techniques: Muscles

Journal: Calcified Tissue International

Article Title: Roles of Insulin-Like Growth Factor-1 in Muscle Wasting and Osteopenia in Mice with Hyponatremia

doi: 10.1007/s00223-025-01369-7

Figure Lengend Snippet: Effects of low sodium concentrations on IGF-1 expression in C2C12 cells. A , B Total RNA was extracted from C2C12 myoblasts ( A ) and myotubes ( B ) 24 or 72 h after culture in medium containing indicated concentrations of sodium, and real-time PCR analysis of IGF-1 and 18S rRNA was performed. Data represent the mean ± SEM (n = 5 sample in each group). Cont; Control. ** P < 0.01 and * P < 0.05

Article Snippet: Mouse myoblastic C2C12 cells (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Wako Pure Chemicals) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: HD6277 Suppresses Muscle Atrophy by Promoting Myogenic Factors and Inhibiting Proteolysis in Aged Mice

doi: 10.1002/jcsm.13805

Figure Lengend Snippet: HD6277 accelerated myotube formation in an Akt‐dependent manner in C2C12 myoblasts. (A,B) The cells were differentiated with various doses of HD6277 (0, 10 or 25 μM) or HD6277 plus GW1100 (10 μM) for 2 days, and then, the phosphorylated Akt level was determined by Western blotting. (C) The cells were differentiated with HD6277 (25 μM) with or without an Akt inhibitor (1 or 2 μM) for 2 days, and then, the transcript level of Mef2c was analysed by qPCR. (D) MyoG and MyHC levels were determined by Western blotting. (E) The MyHC‐positive area and myotube width were calculated in MyHC‐stained cells (green fluorescence). Scale bar: 275 μm. Akti, Akt inhibitor; GW, GW1100; HD, HD6277; Veh, vehicle. Error bars indicate the mean ± SD (* p < 0.05; ANOVA with post hoc t test).

Article Snippet: C2C12 myoblasts and myotubes were stained using a TUNEL assay kit (MyBioSource, San Diego, CA, USA) according to the manufacturer's instructions to visualize DNA fragmentation.

Techniques: Western Blot, Staining, Fluorescence

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: HD6277 Suppresses Muscle Atrophy by Promoting Myogenic Factors and Inhibiting Proteolysis in Aged Mice

doi: 10.1002/jcsm.13805

Figure Lengend Snippet: HD6277 inhibited the palmitate‐mediated reduction in myotube formation in an Akt‐dependent manner in C2C12 myoblasts. (A,B) The cells were stimulated with palmitate (200 μM), palmitate plus HD6277 (10 or 25 μM) or palmitate plus HD6277 with GW1100 (10 μM). The phosphorylated Akt level was analysed by Western blotting. (C) The cells were stimulated with palmitate (200 μM), palmitate plus HD6277 (25 μM) or palmitate plus HD6277 with Akt inhibitor (2 μM) and then differentiated for 2 days. The transcript levels of Mef2c and Myf5 were analysed by qPCR. (D) MyoG and MyHC levels were analysed by Western blotting. (E) MyHC‐stained cells (green fluorescence) were observed under a fluorescence microscope to calculate the MyHC‐positive area and myotube width. Scale bar: 275 μm. Akti, Akt inhibitor; GW, GW1100; HD, HD6277; PA, palmitate; Veh, vehicle. Error bars indicate the mean ± SD (* p < 0.05; ANOVA with post hoc t test).

Article Snippet: C2C12 myoblasts and myotubes were stained using a TUNEL assay kit (MyBioSource, San Diego, CA, USA) according to the manufacturer's instructions to visualize DNA fragmentation.

Techniques: Western Blot, Staining, Fluorescence, Microscopy

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: HD6277 Suppresses Muscle Atrophy by Promoting Myogenic Factors and Inhibiting Proteolysis in Aged Mice

doi: 10.1002/jcsm.13805

Figure Lengend Snippet: HD6277 suppressed palmitate‐mediated cytotoxicity in an Akt‐dependent manner in C2C12 myoblasts. (A,D) The cells were stimulated with palmitate (200 μM), palmitate plus HD6277 (10 or 25 μM) or palmitate plus HD6277 with Akt inhibitor (1 or 2 μM), after which the Bax/Bcl2 ratio and cleaved caspase 3 level were determined by Western blotting. (B,E) Cell viability was measured using the EZ‐Cytox solution. (C,F) DNA fragmentation was visualized using a TUNEL assay kit. Scale bar: 125 μm. TUNEL‐positive cells (red fluorescence) were observed and counted under a fluorescence microscope. Akti, Akt inhibitor; C.Cas‐3, cleaved caspase 3; HD, HD6277; PA, palmitate; Veh, vehicle. Error bars indicate the mean ± SD (* p < 0.05; ANOVA with post hoc t test).

Article Snippet: C2C12 myoblasts and myotubes were stained using a TUNEL assay kit (MyBioSource, San Diego, CA, USA) according to the manufacturer's instructions to visualize DNA fragmentation.

Techniques: Western Blot, TUNEL Assay, Fluorescence, Microscopy

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: HD6277 Suppresses Muscle Atrophy by Promoting Myogenic Factors and Inhibiting Proteolysis in Aged Mice

doi: 10.1002/jcsm.13805

Figure Lengend Snippet: HD6277 reduced the palmitate‐induced proteolytic processes in an Akt‐dependent manner in C2C12 myotubes. (A,B) Fully differentiated cells were stimulated with palmitate (400 μM), palmitate plus HD6277 (10 or 25 μM) or palmitate plus HD6277 with GW1100 (10 μM), and then, the phosphorylated Akt level was determined by Western blotting. (C–F) Fully differentiated cells were stimulated with palmitate (400 μM), palmitate plus HD6277 (10 or 25 μM) or palmitate plus HD6277 with Akt inhibitor (2 μM). Western blotting was then performed to analyse the levels of phosphorylated FOXO1A, atrogin‐1, MuRF1 and ubiquitination. Akti, Akt inhibitor; GW, GW1100; HD, HD6277; PA, palmitate; Veh, vehicle. Error bars indicate the mean ± SD (* p < 0.05; ANOVA with post hoc t test).

Article Snippet: C2C12 myoblasts and myotubes were stained using a TUNEL assay kit (MyBioSource, San Diego, CA, USA) according to the manufacturer's instructions to visualize DNA fragmentation.

Techniques: Western Blot

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: HD6277 Suppresses Muscle Atrophy by Promoting Myogenic Factors and Inhibiting Proteolysis in Aged Mice

doi: 10.1002/jcsm.13805

Figure Lengend Snippet: HD6277 inhibited palmitate‐induced cytotoxicity in an Akt‐dependent manner in C2C12 myotubes. (A,D) Fully differentiated cells were incubated with palmitate (400 μM), palmitate plus HD6277 (10 or 25 μM) or palmitate plus HD6277 with Akt inhibitor (2 μM), and then, Western blotting was performed to determine the levels of the Bax/Bcl2 ratio and cleaved caspase 3. (B,E) Cell viability was measured using EZ‐Cytox solution. (C,F) DNA fragmentation was analysed by the TUNEL assay. Scale bar: 125 μm. TUNEL‐positive cells (red fluorescence) were calculated under a fluorescence microscope. Akti, Akt inhibitor; C.Cas‐3, cleaved caspase 3; HD, HD6277; PA, palmitate; Veh, vehicle. Error bars indicate the mean ± SD (* p < 0.05; ANOVA with post hoc t test).

Article Snippet: C2C12 myoblasts and myotubes were stained using a TUNEL assay kit (MyBioSource, San Diego, CA, USA) according to the manufacturer's instructions to visualize DNA fragmentation.

Techniques: Incubation, Western Blot, TUNEL Assay, Fluorescence, Microscopy

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: HD6277 Suppresses Muscle Atrophy by Promoting Myogenic Factors and Inhibiting Proteolysis in Aged Mice

doi: 10.1002/jcsm.13805

Figure Lengend Snippet: Schematic diagram of HD6277 functions in muscles. The HD6277‐Akt axis enhanced myogenic factors, reduced proteolytic processes and inhibited cytotoxicity in C2C12 cells and muscle tissues from obese and aged mice. Furthermore, HD6277 administration improved muscle strength and the CSA of muscle fibres in aged mice.

Article Snippet: C2C12 myoblasts and myotubes were stained using a TUNEL assay kit (MyBioSource, San Diego, CA, USA) according to the manufacturer's instructions to visualize DNA fragmentation.

Techniques: Muscles