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