Journal: Scientific Reports

Article Title: Repeated intramuscular transplantations of hUCB-MSCs improves motor function and survival in the SOD1 G 93 A mice through activation of AMPK

doi: 10.1038/s41598-020-58221-1

Figure Lengend Snippet: hUCB-MSCs reduced ROS levels induced by TGF-β1 in C2C12 cell myoblasts. ( A ) C2C12 myoblast cells were directly cocultured with hUCB-MSCs with or without TGF-β1. Cocultured myoblast cells were stained with H 2 DCFDA to measure intracellular hydrogen peroxide using FACS caliber. ( B ) To confirm indirect effect of hUCB-MSCs, TGF-β1 treated C2C12 myoblast cells were cocultured with or without hUCB-MSCs and measured intracellular hydrogen peroxide levels. Graphs were plotted as the percentage relative to negative control and analyzed by one-way analysis. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: At 80–90% confluence of cells, media was changed to DMEM containing 1% normal horse serum (NHS, Gibco) to differentiate C2C12 cell myoblasts to myotubes.

Techniques: Staining, Negative Control

Journal: Scientific Reports

Article Title: Repeated intramuscular transplantations of hUCB-MSCs improves motor function and survival in the SOD1 G 93 A mice through activation of AMPK

doi: 10.1038/s41598-020-58221-1

Figure Lengend Snippet: Activation of AMPK by hUCB-MSCs regulated iNOS and protein synthesis in C2C12 cell myotubes. ( A ) C2C12 cell myotubes were treated with TGF-β1 for 24 hr and cocultured with or without hUCB-MSCs and the cell lysates were probed with the indicated antibody. ( B ) The bands of ( A ) were quantified, and the relative expression levels are shown in the graph. ( C ) NO concentration in myotubes treated as described in ( A ) was measured using the Griess assay. ( D ) Representative graph showed relative change in the mRNA expression of SIRT1 and PGC1-alpha. All data were represented as mean ± SEM for three independent experiments and were analyzed by one-way analysis. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: At 80–90% confluence of cells, media was changed to DMEM containing 1% normal horse serum (NHS, Gibco) to differentiate C2C12 cell myoblasts to myotubes.

Techniques: Activation Assay, Expressing, Concentration Assay, Griess Assay

Journal: eLife

Article Title: BRAF activates PAX3 to control muscle precursor cell migration during forelimb muscle development

doi: 10.7554/eLife.18351

Figure Lengend Snippet: ( A ) Immunofluorescence staining of migrating muscle cells (C2C12) after knock down of the hepatocyte growth factor (HGF) receptor (siMet), Braf (siBraf) and Pax3 (siPax3) or after transfection of cultures with Craf, Braf, a dominant negative form of Braf (DN Braf) and CA Braf (V600E) in the presence or absence of HGF. Con indicates control vector and siCon represents a scrambled siRNA control. Cultures were analyzed 4 hr after scratching excluding effects of cell proliferation. Cell numbers were determined by counting the number of DAPI-stained nuclei. A statistical assessment is shown in the upper part of the panel (n = 15; Mann-Whitney-U test, p*<0.05). siRNA knock-down efficiencies for Met (80%), Braf (70%) and Pax3 (85%) were determined by Western blot analysis. ( B ) Phosphorylation of ERK1/2 after transfection of C2C12 cells with control vector (Con), WT Braf and CA Braf (V600E) in the presence or absence of UO126 (5 µM) or cytochalasin D (5 µM; CytoD), noco (5 µM; nocodazole), Dynasore (80 µM; dyna), colchicine (0.01%; colch), methyl-β-cyclodextrin (3 mM; MβCD), and paclitaxel (5 µg/ml; paclit). Addition of DMSO served as an additional control. (n = 2). Cytochalasin D disrupts actin filaments. Nocodazole, colchicine, and paclitaxel interfere with microtubuli assembly or disassembly. U0126 inhibits the MEK/ERK pathway. Dynasore blocks dynamin-dependent endocytosis. Methyl-β-cyclodextrin removes cholesterol from cultured cells and disrupts lipid rafts. ( C ) Statistical assessment of the experiments shown in ( D ) (n = 15; Mann-Whitney-U test, p**<0.01). ( D ) Microscopic imaging of migrating C2C12 cells after transfection with a control vector (Con), WT Braf and CA Braf (V600E) in the presence or absence of DMSO, UO126 and Dynasore. Cultures were analyzed 4 hr after scratching excluding effects of cell proliferation. DOI: http://dx.doi.org/10.7554/eLife.18351.002

Article Snippet: Mass spectrometry was performed after in-gel digestion of Coomassie-stained bands from polyacrylamide gels. ( A ) Proteins identified after GST-PAX3 pull down of protein extracts from WT mouse embryonic forelimbs (E10.5). ( B ) Proteins identified after immunoprecipitation of BRAF (antibody from BD Biosciences (612375)) from extracts of WT mouse embryonic forelimbs (E10.5). ( C ) Proteins identified after immunoprecipitation of PAX3 form C2C12 myoblasts transfected with HA-tagged Pax3. ( D ) Proteins identified after immunoprecipitation of BRAF (antibody from BD Biosciences (612375)) from C2C12 myoblast cells after transfection with HA-tagged Pax3.

Techniques: Immunofluorescence, Staining, Transfection, Dominant Negative Mutation, Plasmid Preparation, MANN-WHITNEY, Western Blot, Cell Culture, Imaging

Journal: eLife

Article Title: BRAF activates PAX3 to control muscle precursor cell migration during forelimb muscle development

doi: 10.7554/eLife.18351

Figure Lengend Snippet: ( A ) Strategy for generation of Braf floxed mice (Braf nfl ). Breeding with MeuCre mice yielded Braf fl mice, in which the neomycin cassette was removed but exon three is flanked by loxP -sites. ( B ) Braf fl mice were bred with Pax3-Cre mice to generate animals lacking Braf in Pax3 expressing cells. Pax3-Cre//Braf del/del embryos die around E15.5. Mutant embryos were analyzed between E10.5 and E14.5. ( C ) RT-PCR analysis of Met and Pax3 expression in limb buds of Pax3-Cre//Braf del/del embryos at E10.5 n = 2. ( D ) Western blot analysis of expression of different markers in limb buds of Pax3-Cre//Braf del/del embryos at E10.5 and E14.5 n = 2. ( E ) Expression of WT and CA Braf (VE600E) in C2C12 cells increases expression of Met and Pax3 . n = 3. ( F ) BRAF enhances PAX3-dependent transcriptional responses. The pGl.3 Pax3BS luc reporter construct containing two PAX3 binding sites in front of a minimal promoter was co-transfected with different combinations of Pax3, Braf, and CA Braf (V600E) expression vectors into HEK293T cells. The activity of Firefly Luciferase was normalized by a co-transfected renilla luciferase in all experiments. Data represent the mean ± SEM and analyzed using ANOVA with a Tukey-Kramer post-hoc comparison test. ***p<0.001. DOI: http://dx.doi.org/10.7554/eLife.18351.004

Article Snippet: Mass spectrometry was performed after in-gel digestion of Coomassie-stained bands from polyacrylamide gels. ( A ) Proteins identified after GST-PAX3 pull down of protein extracts from WT mouse embryonic forelimbs (E10.5). ( B ) Proteins identified after immunoprecipitation of BRAF (antibody from BD Biosciences (612375)) from extracts of WT mouse embryonic forelimbs (E10.5). ( C ) Proteins identified after immunoprecipitation of PAX3 form C2C12 myoblasts transfected with HA-tagged Pax3. ( D ) Proteins identified after immunoprecipitation of BRAF (antibody from BD Biosciences (612375)) from C2C12 myoblast cells after transfection with HA-tagged Pax3.

Techniques: Expressing, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Western Blot, Construct, Binding Assay, Transfection, Activity Assay, Luciferase

Journal: eLife

Article Title: BRAF activates PAX3 to control muscle precursor cell migration during forelimb muscle development

doi: 10.7554/eLife.18351

Figure Lengend Snippet: ( A ) PAX3, GST-PAX3 and PAX3 were immunoprecipitated from protein extracts of E10.5 wild type embryos or from C2C12 muscle cells after transfection with HA-Pax3 or CA Braf (V600E). Immunoprecipitations were analyzed by mass spectrometry after SDS-PAGE and in gel digestions. A selected list of proteins identified by Mascot search analysis is presented. ( B ) Analysis of the interaction of PAX3 and BRAF in E10.5 and E12.5 WT embryos by coupled immunoprecipitation/Western blot analysis. n = 3. ( C ) Western blot analysis of GST and GST-PAX3 immunoprecipitations after transfection of C2C12 cells with empty vector, Braf and CA Braf (V600E). n = 3. ( D ) Western blot analysis of PAX3 or BRAF immunoprecipitations from C2C12 transfected with CA Braf (V600E) after chemical cross-linking (SD) and cleavage of the cross-linker with DTT (SD+DTT). None = no cross-linker added. n = 3. ( E ) Western blot analysis of PAX3 immunoprecipitations from C2C12 transfected with WT Braf, CA Braf (V600E) and vector control (vector). n = 3. DOI: http://dx.doi.org/10.7554/eLife.18351.007

Article Snippet: Mass spectrometry was performed after in-gel digestion of Coomassie-stained bands from polyacrylamide gels. ( A ) Proteins identified after GST-PAX3 pull down of protein extracts from WT mouse embryonic forelimbs (E10.5). ( B ) Proteins identified after immunoprecipitation of BRAF (antibody from BD Biosciences (612375)) from extracts of WT mouse embryonic forelimbs (E10.5). ( C ) Proteins identified after immunoprecipitation of PAX3 form C2C12 myoblasts transfected with HA-tagged Pax3. ( D ) Proteins identified after immunoprecipitation of BRAF (antibody from BD Biosciences (612375)) from C2C12 myoblast cells after transfection with HA-tagged Pax3.

Techniques: Immunoprecipitation, Transfection, Mass Spectrometry, SDS Page, Western Blot, Plasmid Preparation

Journal: eLife

Article Title: BRAF activates PAX3 to control muscle precursor cell migration during forelimb muscle development

doi: 10.7554/eLife.18351

Figure Lengend Snippet: Mass spectrometry was performed after in-gel digestion of Coomassie-stained bands from polyacrylamide gels. ( A ) Proteins identified after GST-PAX3 pull down of protein extracts from WT mouse embryonic forelimbs (E10.5). ( B ) Proteins identified after immunoprecipitation of BRAF (antibody from BD Biosciences (612375)) from extracts of WT mouse embryonic forelimbs (E10.5). ( C ) Proteins identified after immunoprecipitation of PAX3 form C2C12 myoblasts transfected with HA-tagged Pax3. ( D ) Proteins identified after immunoprecipitation of BRAF (antibody from BD Biosciences (612375)) from C2C12 myoblast cells after transfection with HA-tagged Pax3. Selected lists of proteins identified in the different co-immunoprecipitation experiments are shown. DOI: http://dx.doi.org/10.7554/eLife.18351.008

Article Snippet: Mass spectrometry was performed after in-gel digestion of Coomassie-stained bands from polyacrylamide gels. ( A ) Proteins identified after GST-PAX3 pull down of protein extracts from WT mouse embryonic forelimbs (E10.5). ( B ) Proteins identified after immunoprecipitation of BRAF (antibody from BD Biosciences (612375)) from extracts of WT mouse embryonic forelimbs (E10.5). ( C ) Proteins identified after immunoprecipitation of PAX3 form C2C12 myoblasts transfected with HA-tagged Pax3. ( D ) Proteins identified after immunoprecipitation of BRAF (antibody from BD Biosciences (612375)) from C2C12 myoblast cells after transfection with HA-tagged Pax3.

Techniques: Mass Spectrometry, Staining, Immunoprecipitation, Transfection

Journal: eLife

Article Title: BRAF activates PAX3 to control muscle precursor cell migration during forelimb muscle development

doi: 10.7554/eLife.18351

Figure Lengend Snippet: ( A ) Western blot analysis of cytoplasmic (C) and nuclear fractions (N) of C2C12 cells transfected with CA Braf (V600E), WT Braf, HA-Pax3 or HA-Pax3-GFP. n = 3. Successful fractionation was monitored by cytoplasmic GAPDH and the nuclear protein LAMIN A/C. Some cultures were treated with Dynasore (Dyna) for 30 min before fractionation as indicated. ( B ) Western blot analysis of immunoprecipitations of nuclear fractions isolated from migrating C2C12 cells after transfection with WT Braf, CA Braf (V600E), or HA-Pax3. n = 3. ( C ) High resolution confocal images of C2C12 cells transfected with WT, CA Braf (V600E) and HA-Pax3-GFP. ( D ) High resolution confocal image of a PAX3 and BRAF positive forelimb muscle precursor cell at E10.5 (left panel). A lower magnification is shown in the right panel. DOI: http://dx.doi.org/10.7554/eLife.18351.009

Article Snippet: Mass spectrometry was performed after in-gel digestion of Coomassie-stained bands from polyacrylamide gels. ( A ) Proteins identified after GST-PAX3 pull down of protein extracts from WT mouse embryonic forelimbs (E10.5). ( B ) Proteins identified after immunoprecipitation of BRAF (antibody from BD Biosciences (612375)) from extracts of WT mouse embryonic forelimbs (E10.5). ( C ) Proteins identified after immunoprecipitation of PAX3 form C2C12 myoblasts transfected with HA-tagged Pax3. ( D ) Proteins identified after immunoprecipitation of BRAF (antibody from BD Biosciences (612375)) from C2C12 myoblast cells after transfection with HA-tagged Pax3.

Techniques: Western Blot, Transfection, Fractionation, Isolation

Journal: eLife

Article Title: BRAF activates PAX3 to control muscle precursor cell migration during forelimb muscle development

doi: 10.7554/eLife.18351

Figure Lengend Snippet: ( A ) Immunofluorescence images of C2C12 cells in a Boyden micro chemotaxis chamber. Cultures were either not stimulated (Con) or treated with HGF after transfection with CA Braf (V600E). Note: Overlay of red BRAF and green PAX3 results in a yellow color of the nuclei. ( B ) High resolution confocal images of nuclei of migrating C2C12 cells transfected with WT and CA Braf (V600E). Cells were co-stained for P-BRAF and EEA1 as well as for P-BRAF and PAX3. Nuclei were visualized by DAPI. Note the absence of the endosomal marker EEA1 in nuclei, while P-BRAF is clearly visible within nuclei. DOI: http://dx.doi.org/10.7554/eLife.18351.010

Article Snippet: Mass spectrometry was performed after in-gel digestion of Coomassie-stained bands from polyacrylamide gels. ( A ) Proteins identified after GST-PAX3 pull down of protein extracts from WT mouse embryonic forelimbs (E10.5). ( B ) Proteins identified after immunoprecipitation of BRAF (antibody from BD Biosciences (612375)) from extracts of WT mouse embryonic forelimbs (E10.5). ( C ) Proteins identified after immunoprecipitation of PAX3 form C2C12 myoblasts transfected with HA-tagged Pax3. ( D ) Proteins identified after immunoprecipitation of BRAF (antibody from BD Biosciences (612375)) from C2C12 myoblast cells after transfection with HA-tagged Pax3.

Techniques: Immunofluorescence, Chemotaxis Assay, Transfection, Staining, Marker

Journal: eLife

Article Title: BRAF activates PAX3 to control muscle precursor cell migration during forelimb muscle development

doi: 10.7554/eLife.18351

Figure Lengend Snippet: ( A ) Western blot analysis of isolated subcellular fractions of C2C12 cells after transfection of CA Braf V600E and knockdown of Eea1 or Erk1/2 . n = 3. Knockdown of Eea1 prevented accumulation of BRAF in the nucleus (N). C: cytoplasm. siRNA knock-down efficiencies for Eea1 (60%) and Erk (95%) were determined by Western blot analysis. ( B ) Knock down of Braf (siBRAF) but not of Eea1 (siEea1), Dnm-2 (siDnm2), Cltc (siCltc), Cav1 (siCav1) and Arf6 (siArf6) did prevent phosphorylation of ERK1/2. Western blot analyses of siRNA transfected C2C12 cells are shown. n = 3. Actin served as loading control. siRNA knock-down efficiencies for Pax3 (85%), Braf (70%), Arf6 (80%), Cav1 (75%), Cltc (70%), Dnm2 (75%) and Eea1 (60%) were determined by Western blot analysis. ( C ) Immunofluorescence staining of migrating C2C12 cells after knockdown of Eea1 or Erk-1/2 . Cultures were transfected with control vector, Braf or CA Braf (V600E) as indicated. Con indicates control cultures and siCon means control siRNA. siRNA knock-down efficiencies for Eea1 (60%) and Erk (95%) were determined by Western blot analysis. Cell numbers were determined by counting the number of DAPI-stained nuclei. A statistical analysis of siEea1 versus siCon in CA Braf (VE600E) transfected cultures is shown. n = 12; Mann-Whitney-U test, (p*<0.05). DOI: http://dx.doi.org/10.7554/eLife.18351.011

Article Snippet: Mass spectrometry was performed after in-gel digestion of Coomassie-stained bands from polyacrylamide gels. ( A ) Proteins identified after GST-PAX3 pull down of protein extracts from WT mouse embryonic forelimbs (E10.5). ( B ) Proteins identified after immunoprecipitation of BRAF (antibody from BD Biosciences (612375)) from extracts of WT mouse embryonic forelimbs (E10.5). ( C ) Proteins identified after immunoprecipitation of PAX3 form C2C12 myoblasts transfected with HA-tagged Pax3. ( D ) Proteins identified after immunoprecipitation of BRAF (antibody from BD Biosciences (612375)) from C2C12 myoblast cells after transfection with HA-tagged Pax3.

Techniques: Western Blot, Isolation, Transfection, Immunofluorescence, Staining, Plasmid Preparation, MANN-WHITNEY

Journal: eLife

Article Title: BRAF activates PAX3 to control muscle precursor cell migration during forelimb muscle development

doi: 10.7554/eLife.18351

Figure Lengend Snippet: ( A ) Exchange of serine by alanine in PAX3 at Ser205 reduces C2C12 myoblast migration after transfection with CA Braf (V600E). Cultures were either transfected with a control vector (-), with wildtype Pax3 (WT) or with mutated Pax3 constructs. Mutated serine residues are indicated (S201A, S205A, S209A, S222A). Cell numbers were determined by counting the number of DAPI-stained nuclei. n = 10; Mann-Whitney-U test, (p*<0.05). ( B ) Phosphorylated PAX3 proteins were isolated from Pax3 and mutant Braf (V600E) transfected HEK293T or C2C12 cells by phospho-column affinity purification and detected by immunoblotting. Phosphorylated ERK1/2 (P-ERK1/2), ERK2 and EZRIN served as controls. Two sets of independent experiments are shown for HEK293T cells. Molecular sizes (in kDa) are indicated by the overlay of the colored marker with the PAX3 and BRAF bands visualized by chemiluminescence. Note that PAX3 shows a shift in the molecular weight due to the HA-tag. Stained membranes with marker are shown. ( C ) In vitro kinase assay of GST, GST-PAX3, PAX3 and different PAX3 mutants demonstrating phosphorylation of PAX3 by BRAF. Bacterially produced GST-PAX3 (WT and mutants) was purified by GST-affinity chromatography. Column eluates and inputs were incubated in vitro together with BRAF. n = 2. Phosphorylated PAX3 was detected using an antibody specific for p-Ser205 PAX3. Specificity of the p-Ser205 PAX3 antibody was assessed by the failure to detect the S205A PAX3 mutant (bottom panel). DOI: http://dx.doi.org/10.7554/eLife.18351.014

Article Snippet: Mass spectrometry was performed after in-gel digestion of Coomassie-stained bands from polyacrylamide gels. ( A ) Proteins identified after GST-PAX3 pull down of protein extracts from WT mouse embryonic forelimbs (E10.5). ( B ) Proteins identified after immunoprecipitation of BRAF (antibody from BD Biosciences (612375)) from extracts of WT mouse embryonic forelimbs (E10.5). ( C ) Proteins identified after immunoprecipitation of PAX3 form C2C12 myoblasts transfected with HA-tagged Pax3. ( D ) Proteins identified after immunoprecipitation of BRAF (antibody from BD Biosciences (612375)) from C2C12 myoblast cells after transfection with HA-tagged Pax3.

Techniques: Migration, Transfection, Plasmid Preparation, Construct, Staining, MANN-WHITNEY, Isolation, Mutagenesis, Affinity Column, Purification, Western Blot, Marker, Molecular Weight, In Vitro, Kinase Assay, Produced, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Eburicoic Acid, a Triterpenoid Compound from Antrodia camphorata, Displays Antidiabetic and Antihyperlipidemic Effects in Palmitate-Treated C2C12 Myotubes and in High-Fat Diet-Fed Mice

doi: 10.3390/ijms18112314

Figure Lengend Snippet: Effects of eburicoic acid (TRR) on the insulin (Ins)-stimulated expression levels of membrane glucose transporter type 4 (GLUT4), the ratio of phospho-5′-adenosine monophosphate kinase (p-AMPK) to total AMPK (t-AMPK), or phospho-Akt (p-Akt)/total Akt (t-Akt) in insulin-resistant C2C12 myotube cells induced by palmitate (Pal). The symbols “+++”, “###” and “***” represent p < 0.001 as respectively compared to the value of the blank control, positive control (insulin) and negative control (insulin + palmitate) using analysis of variance (ANOVA) and with Dunnett’s tests. ( A ) Representative image. ( B – D ) Quantification of the membrane GLUT4 expression levels, the ratio of p-AMPK to t-AMPK, or p-Akt/t-Akt expression levels. CON: Blank control; DMSO: Dimethyl sulfoxide, solvent control.

Article Snippet: Myoblast C2C12 cells that were not treated with the TRR, dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St Loiuis, MO, USA), insulin, and palmitate served as a blank control (as the control group).

Techniques: Expressing, Positive Control, Negative Control