Journal: Current Issues in Molecular Biology

Article Title: Caprylic Acid Restores Branched-Chain Amino Acid Metabolism in a Mouse Cachexia Model

doi: 10.3390/cimb47050325

Figure Lengend Snippet: Effect of BCAAs on energy metabolism in an in vitro cachexia model. C2C12 cells (1 × 10 4 ) cells were pretreated with cancer ascites (20% v / v ) to fresh DMEM medium. Then, cells were cultured in the mitochondrial stress test medium for 6 h. ( A ) Oxidative phosphorylation by flux analysis. ( B ) Basal respiration. ( C ) Maximum respiration. ( D ) ATP production. ( E ) Proton leak. ( F ) Spare respiration capacity. ( G ) ECAR. ( H ) Maximum ECAR. Error bar, standard deviation from 3 independent trials. Statistical differences were calculated by ordinary ANOVA with Bonferroni’s correction. ANOVA, analysis of variance; Asc, cancer ascites; ATP, adenosine triphosphate; BCAA, branched-chain amino acid; C, control; DMEM, Dulbecco’s Modified Eagle’s Medium; ECAR, Glycolysis by extracellular acidity rate; OCR, oxygen consumption rates.

Article Snippet: C2C12 myoblasts derived from mouse skeletal muscle were obtained from Dainihon Pharmacy Co. (Tokyo, Japan).

Techniques: In Vitro, Cell Culture, Phospho-proteomics, Standard Deviation, Control, Modification

Journal: Current Issues in Molecular Biology

Article Title: Caprylic Acid Restores Branched-Chain Amino Acid Metabolism in a Mouse Cachexia Model

doi: 10.3390/cimb47050325

Figure Lengend Snippet: Impaired BCAA metabolism in an in vitro cachexia model. ( A – C ) C2C12 cells (1 × 10 6 ) were treated with cancer ascites (20% v / v to fresh DMEM medium) with or without BCAAs (Leu, Ile, and Val, 200 μM each) for 48 h. ( A ) Levels of BCAA metabolism-associated proteins. Right panel: semi-quantification of Western blotting. ( B ) Intramuscular BCAA concentration. ( C ) Intramuscular AcCoA concentration. ( D – H ) C2C12 cells were treated with HMGB1 (40 μg/mL) with or without BCAAs (Leu, Ile, and Val, 200 μM each) or C8 (50 μg/mL) for 48 h. ( D ) Expression of BCKD-related genes. Right panel: semi-quantification of RT-PCR. ( E , F ) Effect of HMGB1 on intramuscular BCAA ( E ) and AcCoA ( F ) concentrations. ( G , H ) Effect of C8 on intramuscular BCAA ( G ) and AcCoA ( H ) concentrations. Error bar: standard deviation from 3 independent trials. Statistical differences were calculated by ordinary ANOVA with Bonferroni’s correction. AcCoA, acetyl coenzyme A; ACTB, β-actine; ANOVA, analysis of variance; Asc, cancer ascites; BCAA, branched-chain amino acid; BDK, branched-chain ketoacid dehydrogenase kinase; BCKD, branched-chain α-ketoacid dehydrogenase; C8, caprylic acid; Cont, control; C, control; DMEM, Dulbecco’s Modified Eagle’s Medium; HMGB1, high-mobility group box-1; pBCKD, phosphorylated; PGC1A, peroxisome proliferator-activated receptor-γ coactivator-1α; RT-PCR, reverse transcription—polymerase chain reaction.

Article Snippet: C2C12 myoblasts derived from mouse skeletal muscle were obtained from Dainihon Pharmacy Co. (Tokyo, Japan).

Techniques: In Vitro, Western Blot, Concentration Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Standard Deviation, Control, Modification, Reverse Transcription, Polymerase Chain Reaction

Journal: Current Issues in Molecular Biology

Article Title: Caprylic Acid Restores Branched-Chain Amino Acid Metabolism in a Mouse Cachexia Model

doi: 10.3390/cimb47050325

Figure Lengend Snippet: Effect of BCAAs combined with HMGB1 and/or C8 on energy metabolism in C2C12 cells. C2C12 cells were treated with BCAAs (Leu, Ile, and Val, 200 μM each), HMGB1 (40 μg/mL), and/or C8 (50 μg/mL) for 48 h. For flux assay, cells were then cultured in the mitochondrial stress test medium for 6 h. ( A ) Oxidative phosphorylation by flux analysis. ( B ) Basal respiration. ( C ) Maximum respiration. ( D ) ATP production. ( E ) Proton leak. ( F ) Maximum ECAR. ( G ) SDS-MYL1. ( H ) 4HNE. Error bar: standard deviation from 3 independent trials. Statistical differences were calculated by ordinary ANOVA with Bonferroni’s correction. 4HNE, 4-hydroxynonenal; ANOVA, analysis of variance; ATP, adenosine triphosphate; BCAA, branched-chain amino acid; C, control; C8, caprylic acid; ECAR, extracellular acidity rate; HMGB; HMGB1, high-mobility group box-1; OCR, oxygen consumption rates; SDS-MYL1, SDS-soluble myosin light chain-1.

Article Snippet: C2C12 myoblasts derived from mouse skeletal muscle were obtained from Dainihon Pharmacy Co. (Tokyo, Japan).

Techniques: Flux Assay, Cell Culture, Phospho-proteomics, Standard Deviation, Control

Journal: Cellular and Molecular Life Sciences

Article Title: Notch1 signaling is mediated by importins alpha 3, 4, and 7

doi: 10.1007/s00018-010-0378-7

Figure Lengend Snippet: NICD in vitro binds to importins α3, α4, and α7. a GST pull-down assays were performed with lysate of HEK293 cells stably transfected with NotchΔE and purified recombinant GST-importins as indicated. Proteins were separated on SDS-PAGE, blotted and labeled with NICD-specific antibody ( top ) or stained with Coomassie Brilliant Blue ( bottom ). b Using purified recombinant GST-NICD, pull-down assays were performed from lysates of C2C12 cells ( left ) or mouse skeletal muscle ( right ). Lysates, pull-down, and as controls pull-down with GST protein and GSH-beads were separated on SDS-PAGE, blotted, and labeled with importin-specific antibodies as indicated ( top , WB) or gels were stained with Coomassie Brilliant Blue ( bottom ). Asterisk , unspecific bands; WB , Western blot. c For co-immunoprecipitation experiment (Co-IP) C2C12 cell lysate transiently transfected with NICD-myc was immunoprecipitated with anti-importin α4 antibody or normal goat immunoglobulins. Lysate and Co-IPs were separated on SDS-PAGE, blotted, and labeled with importin α4- and myc-specific antibodies

Article Snippet: HeLa Kyoto cells and mouse myoblast C2C12 cells were kindly provided by Rainer Pepperkok (EMBL, Heidelberg) and Rüdiger Rudolf (Forschungszentrum Karlsruhe, Eggenstein-Leopoldshafen), respectively.

Techniques: In Vitro, Stable Transfection, Transfection, Purification, Recombinant, SDS Page, Labeling, Staining, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Cellular and Molecular Life Sciences

Article Title: Notch1 signaling is mediated by importins alpha 3, 4, and 7

doi: 10.1007/s00018-010-0378-7

Figure Lengend Snippet: Endogenous Notch signaling in myoblasts is mainly mediated by importins α3 and α4. a C2C12 cells transfected with siRNAs against importin isoforms as indicated were transfected with or without Delta1 cDNA and Notch reporter construct. The γ-secretase inhibitor DAPT was used to show γ-secretase dependency of the measured Notch activity. Firefly/renilla activities were determined and the activity in Delta1 transfected cells set to 100%. Means ± SD of five independent experiments are shown. Asterisks indicate significance ( p < 0.05, Student's t test). b Western-blot analysis of importin KD efficiency and specificity. C2C12 cell lysates were separated on SDS-PAGE, blotted, and probed with antibodies as indicated. c C2C12 cells were transfected with control (ctrl) siRNA or pooled siRNAs against importins α3, α4, and α7 and subsequently with or without Delta1 as indicated. Where indicated, cells were incubated with DAPT for 24 h. After RNA isolation, quantitative real-time PCR for Hey1 expression was performed. Hey1 expression level after Delta1 induction was set to 100% and the other values related to that. Means ± SD of three independent experiments are shown

Article Snippet: HeLa Kyoto cells and mouse myoblast C2C12 cells were kindly provided by Rainer Pepperkok (EMBL, Heidelberg) and Rüdiger Rudolf (Forschungszentrum Karlsruhe, Eggenstein-Leopoldshafen), respectively.

Techniques: Transfection, Construct, Activity Assay, Western Blot, SDS Page, Control, Incubation, Isolation, Real-time Polymerase Chain Reaction, Expressing

Journal: Cell Death & Disease

Article Title: TRAIL/DR5 pathway promotes AKT phosphorylation, skeletal muscle differentiation, and glucose uptake

doi: 10.1038/s41419-021-04383-3

Figure Lengend Snippet: Experiments were conducted in C2C12 cells on D3 (3 rd day of differentiation), after 24 h exposure to oleic acid or palmitic acid ± TRAIL. A , B Cell viability. C , D Quantification of lipid droplet content (expressed as percentage of Oil red O stained area). E Representative images of C2C12 cells stained with DAPI (left panels) and LipidTOX (right panels) in presence or absence of oleic acid (500 µM) ± TRAIL (100 ng/mL), scale bar 50 μm. F , G Gene expression of Pparg , Pcg1α , Cpt1b , Pepck1, Myod , Myog , and Myh4 . Gene expression is reported as mRNA fold induction normalized to the mRNA level of CNT. H Fusion index quantification. The fusion index corresponds to the ratio between the number of nuclei in a MyHC-positive myotube (with > 3 nuclei) and the total number of nuclei. I Representative images of C2C12 cells stained with MitoSox (red staining, top panels) and anti-MyHC (green staining, bottom panels) in the presence or absence of oleic acid (500 µM) ± TRAIL (10 ng/mL), scale bar 50 μm. Results were obtained by 3–5 independent experiments and are presented as median with interquartile range. Significance was assessed with ANOVA and Tukey.

Article Snippet: Mouse C2C12 myoblasts (ECACC General Cell Collection, Merck, Darmstadt, Germany) were cultured as previously described [ ].

Techniques: Staining, Gene Expression

Journal: Cell Death & Disease

Article Title: TRAIL/DR5 pathway promotes AKT phosphorylation, skeletal muscle differentiation, and glucose uptake

doi: 10.1038/s41419-021-04383-3

Figure Lengend Snippet: Experiments were conducted in C2C12 cells during their differentiation. TRAIL treatment was renewed every 24 h between D0 and D4 (D is for day of differentiation). A – B Densitometric analysis and C representative blots of myogenin and MyHC protein expression. D Myotube length. E Fusion index. F Representative images of C2C12 cell morphology. Brightfield (upper panels), MyHC immunofluorescence (middle panels), DAPI staining (lower panels). Scale bar represents 100 µm. G – J Gene expression of Myog , Glut4 , Pepck1 , and Pgk1 . Gene expression is reported as mRNA fold induction normalized to the mRNA level of CNT cells on D1. Results were obtained by 3–4 independent experiments and are presented as median with interquartile range. Significance was assessed with ANOVA and Tukey.

Article Snippet: Mouse C2C12 myoblasts (ECACC General Cell Collection, Merck, Darmstadt, Germany) were cultured as previously described [ ].

Techniques: Expressing, Immunofluorescence, Staining, Gene Expression

Journal: Cell Death & Disease

Article Title: TRAIL/DR5 pathway promotes AKT phosphorylation, skeletal muscle differentiation, and glucose uptake

doi: 10.1038/s41419-021-04383-3

Figure Lengend Snippet: Experiments were conducted in C2C12 cells during their differentiation (D0–D3). Dr5 was silenced before D0. A , C , E , G Gene expression of Dr5 , Myod , Myog, and Myh4 . Gene expression is reported as mRNA fold induction normalized to siCTR mRNA level on D0. B , D , F , H Quantification of DR5, MyoD, myogenin, and MyHC protein expression, and I representative blots. J Quantification of myotube length and K the fusion index on D3. Results were obtained by 3–4 independent experiments and are presented as median with interquartile range. Significance was assessed with ANOVA and Tukey tests ( A – H ), and J , K t -test. L Representative images of C2C12 cell morphology after Dr5 silencing, as assessed on D3. Images are presented in brightfield, and/or after anti-DR5 staining (green staining), and/or after anti-MyHC staining (red staining). Scale bar = 50 µm.

Article Snippet: Mouse C2C12 myoblasts (ECACC General Cell Collection, Merck, Darmstadt, Germany) were cultured as previously described [ ].

Techniques: Gene Expression, Expressing, Staining

Journal: Cell Death & Disease

Article Title: TRAIL/DR5 pathway promotes AKT phosphorylation, skeletal muscle differentiation, and glucose uptake

doi: 10.1038/s41419-021-04383-3

Figure Lengend Snippet: Experiments were conducted in C2C12 cells during their differentiation (D0–D3). TRAIL treatment was renewed every 24 h. A Representative blots and B – D quantification of LC3B-I and LC3B-II, p62, and myogenin expression on D3. E – F Representative blots and quantification of AMPKα and P-AMPKα expression on D3. G – H Representative blots and quantification of AKT and P-AKT expression on D3. I – J Representative blots and quantification of DR5, AKT, and P-AKT after Dr5 silencing on D3. K – L Representative blots and quantification of AKT and P-AKT in C2C12 cells treated with TRAIL (24 h on D3) ± insulin (20 min on D5 after serum starvation). M Glucose uptake in C2C12 cells treated with TRAIL (24 h on D3) ± insulin (20 min on D5 after serum starvation). Results were obtained by 3–4 independent experiments and are presented as median with interquartile range. Significance was assessed with ANOVA followed by Tukey test, except for J where it was used the t -test.

Article Snippet: Mouse C2C12 myoblasts (ECACC General Cell Collection, Merck, Darmstadt, Germany) were cultured as previously described [ ].

Techniques: Expressing

Journal: Burns & Trauma

Article Title: Enhancing diabetic muscle repair through W-GA nanodots: a nanomedicinal approach to ameliorate myopathy in type 2 diabetes

doi: 10.1093/burnst/tkae059

Figure Lengend Snippet: Evaluation of the biocompatibility of W-GA in vitro. ( a ) C2C12 cells cultured for 7 days in W-GA were evaluated for proliferation and cytotoxicity using live/dead staining. Scale bar = 500 μm. ( b ) Survival rate of C212 cells ( n = 3) is shown as the mean ± standard deviation. The statistical significance of differences between treatments was determined by one-way ANOVA and the Bonferroni posthoc correction. NS: Not significant. ( c ) C2C12 cell proliferation capacity was assessed 24 hours post-treatment using BrdU incorporation. The green signal represents BrdU. Scale bar = 500 μm. ( d ) Quantification of BrdU assay data ( n = 3). The data are presented as the mean ± standard deviation. NS: Not significant. ( e ) The cell proliferation ability of C2C12 cells in the W-GA group was further evaluated using a CCK-8 assay. The data are presented as the mean ± standard deviation. NS: Not significant

Article Snippet: C2C12 myoblasts (a mouse cell line) were obtained from ScienCell Research Laboratories and cultured in Dulbecco's modified Eagle’s medium (DMEM; containing 25 mM glucose; Gibco, USA), which included 10% fetal bovine serum (Gibco, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, USA), in a humidified atmosphere at 37°C and 5% CO 2 .

Techniques: In Vitro, Cell Culture, Staining, Standard Deviation, BrdU Incorporation Assay, BrdU Staining, CCK-8 Assay

Journal: Burns & Trauma

Article Title: Enhancing diabetic muscle repair through W-GA nanodots: a nanomedicinal approach to ameliorate myopathy in type 2 diabetes

doi: 10.1093/burnst/tkae059

Figure Lengend Snippet: Antiapoptotic, antioxidative, and myogenic differentiation-promoting effects of W-GA. ( a ) Flow cytometry profiles showing the abundance of total C2C12 cells under various treatment conditions, along with apoptosis events in C2C12 cells under different therapeutic interventions. ( b ) Quantification of flow cytometry data for apoptotic cells ( n = 3). The data are presented as the mean ± standard deviation. NS: Not significant, * * p < 0.01. The statistical significance of differences between treatments was determined by one-way ANOVA and the Bonferroni posthoc correction. ( c ) Flow cytometry profiles showing the production of ROS in C2C12 cells under different treatment conditions. ( d ) Quantification of flow cytometry data for ROS production ( n = 3). The data are presented as the mean ± standard deviation. NS: Not significant, * p < 0.05. ( e ) Representative immunofluorescence image illustrating MYHC and MyoD protein expression in C2C12 myoblasts. Scale bar = 100 μm. ( f ) Quantitative analysis and intergroup comparison of myotube diameters ( n = 3). The statistical significance of differences between treatments was determined by one-way ANOVA and the Bonferroni posthoc correction. NS: Not significant, * p < 0.05, * * p < 0.01

Article Snippet: C2C12 myoblasts (a mouse cell line) were obtained from ScienCell Research Laboratories and cultured in Dulbecco's modified Eagle’s medium (DMEM; containing 25 mM glucose; Gibco, USA), which included 10% fetal bovine serum (Gibco, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, USA), in a humidified atmosphere at 37°C and 5% CO 2 .

Techniques: Flow Cytometry, Standard Deviation, Immunofluorescence, Expressing, Comparison