Journal: PLoS ONE

Article Title: Alpha1a-Adrenoceptor Genetic Variant Triggers Vascular Smooth Muscle Cell Hyperproliferation and Agonist Induced Hypertrophy via EGFR Transactivation Pathway

doi: 10.1371/journal.pone.0142787

Figure Lengend Snippet: A-10 SMCs stably expressing control plasmid, WT or 247R receptors were cultured in (A) 0.5% FBS or (B) 10% FBS-containing medium for 48h in the presence or absence of agonist (A61603 or PE), or (C) in the presence or absence of 1μM prazosin (α 1 AR inverse agonist). Cell proliferation was evaluated by cell counting using light microscopy. Data represent mean ± SE of 3 independent experiments each performed in triplicates and analyzed by two-way ANOVA followed by post-hoc Tukey’s test.

Article Snippet: A-10 embryonic rat aorta thoracic/medial layer-derived SMCs (ATCC ® CRL-1476, Manassas, VA) were cultured in DMEM (Gibco, Auckland, NZ) supplemented with 10% FBS (Hyclone Laboratories, South Logan, UT) and 100U/ml penicillin/streptomycin (Gibco, Grand Island, NY) at 37°C in 5% CO 2 .

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Cell Culture, Cell Counting, Light Microscopy

Journal: PLoS ONE

Article Title: Alpha1a-Adrenoceptor Genetic Variant Triggers Vascular Smooth Muscle Cell Hyperproliferation and Agonist Induced Hypertrophy via EGFR Transactivation Pathway

doi: 10.1371/journal.pone.0142787

Figure Lengend Snippet: (A) A-10 SMCs expressing WT or 247R receptors were cultured in 0.5% FBS-containing medium in the presence or absence of AG1478 (EGFR-specific inhibitor) or GM6001 (general MMP inhibitor) or (B) either PD98059 or UO126 MEK inhibitors or (C) agonist for 48h. Cell lysates were analyzed by Western blotting and cell proliferation was evaluated by light microscopy. Lower panels depict relative intensities of phospho-protein bands as determined by densitometric analysis. Data represent mean ± SE of 5 (A), 4 (B) or 3 (C) independent experiments and analyzed by two-way ANOVA followed by post-hoc Tukey’s test.

Article Snippet: A-10 embryonic rat aorta thoracic/medial layer-derived SMCs (ATCC ® CRL-1476, Manassas, VA) were cultured in DMEM (Gibco, Auckland, NZ) supplemented with 10% FBS (Hyclone Laboratories, South Logan, UT) and 100U/ml penicillin/streptomycin (Gibco, Grand Island, NY) at 37°C in 5% CO 2 .

Techniques: Expressing, Cell Culture, Western Blot, Light Microscopy

Journal: PLoS ONE

Article Title: Alpha1a-Adrenoceptor Genetic Variant Triggers Vascular Smooth Muscle Cell Hyperproliferation and Agonist Induced Hypertrophy via EGFR Transactivation Pathway

doi: 10.1371/journal.pone.0142787

Figure Lengend Snippet: (A) Control A-10 SMCs, or cells expressing WT or 247R receptors were cultured for 48h in 0.5% FBS-containing medium in the presence or absence of 10μM PE, followed by cell membrane staining with wheat germ agglutinin. Average cell surface area was evaluated by Image J software at 20x magnification. (B,C) Cells were cultured for 48h in 0.5% FBS-containing medium in the presence or absence of EGFR-specific inhibitor AG1478, MMP inhibitor GM6001 and/or 10μM PE. Cell lysates were analyzed by Western blotting. Data represent mean ± SE of 3 (A, C) or 4 (B) independent experiments and analyzed by one-way ANOVA followed by post-hoc Tukey’s test (A) or two-way ANOVA followed by post-hoc Tukey’s (B) or Bonferroni (C) tests.

Article Snippet: A-10 embryonic rat aorta thoracic/medial layer-derived SMCs (ATCC ® CRL-1476, Manassas, VA) were cultured in DMEM (Gibco, Auckland, NZ) supplemented with 10% FBS (Hyclone Laboratories, South Logan, UT) and 100U/ml penicillin/streptomycin (Gibco, Grand Island, NY) at 37°C in 5% CO 2 .

Techniques: Expressing, Cell Culture, Staining, Software, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Protein Kinase A-regulated Assembly of a MEF2·HDAC4 Repressor Complex Controls c-Jun Expression in Vascular Smooth Muscle Cells

doi: 10.1074/jbc.m109.000539

Figure Lengend Snippet: FIGURE 1. MEF2 activity and expression in VSMCs in vivo and in vitro. Common carotid arteries of MEF2-LacZ mice were injured by inserting a 2-mm wire into the external carotid. Contralateral arteries were used as the control. A, X-gal staining and immunofluorescence (IF) for MEF2A and c-Jun 14 days following injury. B, A10 cells were transfected with the wild-type c-jun promoter (c-Jun-luc). Following recovery, the cells were serum-starved overnight and treated with 20% FBS, 100 M H2O2, 60 mM KCl, or 10 ng/ml of PDGF for 4 h for luciferase extracts or 2 h for protein extracts. C, growth phase A10s in 10% FBS () or serum-free medium () were harvested for protein subjected to immunoblotting (IB) for MEF2A, MEF2C, and MEF2D. D, A10 cells were transfected with the c-jun or MEF2 reporter genes and MEF2A or MEF2A-VP16 (*, p 0.05 was considered statistically significant).

Article Snippet: Cell Culture and Treatment of VSMCs—Rat A10 myoblasts (ATCC; CRL-1476) were maintained in growth medium consisting of 10% fetal bovine serum (FBS).

Techniques: Activity Assay, Expressing, In Vivo, In Vitro, Control, Staining, Immunofluorescence, Transfection, Luciferase, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Protein Kinase A-regulated Assembly of a MEF2·HDAC4 Repressor Complex Controls c-Jun Expression in Vascular Smooth Muscle Cells

doi: 10.1074/jbc.m109.000539

Figure Lengend Snippet: FIGURE 2. PDGF induction of c-Jun is mediated by CaMK, PKC, and MEK. A, serum-starved A10 cells were treated with PDGF (10 ng/ml) for 2 h following 15 min pretreatment with rottlerin (5 M), KN-62 (5 M), LY294002 (10 M), or PD98059 (10 M). Protein extracts were immunoblotted (IB) with a c-Jun antibody (H79, Santa Cruz). B, growth phase VSMCs were treated with rottlerin (5 M) and KN-62 (5 M) followed by fixation with 4% paraformaldehyde. The fixed cells were then subjected to immunofluorescence (IF) with an HDAC4 primary antibody (Sigma). C, VSMCs were transfected with an EGFP fusion protein containing full-length human HDAC4 (HDAC4-GFP), and either activated CaMKII deltaB, CaMKIV, or PKD. Following serum starvation micrographs were obtained by standard fluorescent techniques. D, A10s were transfected with HDAC4-GFP. Micrographs were obtained in serum-free medium, low density growth medium (10% FBS), and high density growth medium (10% FBS). E, nuclear and cytosolic extracts were made from cultured VSMCs treated with 10 ng/ml PDGF for 2 h. The extracts were subjected to SDS-PAGE and immunoblotted for HDAC4, MEF2D, or GAPDH.

Article Snippet: Cell Culture and Treatment of VSMCs—Rat A10 myoblasts (ATCC; CRL-1476) were maintained in growth medium consisting of 10% fetal bovine serum (FBS).

Techniques: Immunofluorescence, Transfection, Cell Culture, SDS Page

Journal: Journal of Biological Chemistry

Article Title: Protein Kinase A-regulated Assembly of a MEF2·HDAC4 Repressor Complex Controls c-Jun Expression in Vascular Smooth Muscle Cells

doi: 10.1074/jbc.m109.000539

Figure Lengend Snippet: FIGURE 3. HDAC4 expression in models of vascular disease. A, A10 cells were grown to confluence and scraped with a standard 200-l pipette tip. The cells were re-fed either serum-free medium or medium con- taining10%FBSovernightandthenfixedforimmunofluorescence.Red,HDAC4;green,4,6-diamidino-2-phen- ylindole (i.e. nuclear). B, Sprague-Dawley rats were subjected to balloon-injury of the carotid artery. Following 14 days of recovery, the arteries were fixed and harvested for immunofluorescence. Green, HDAC4; blue, To- pro-3 (i.e. nuclear). C, human aortic aneurysms or a nondiseased control specimen were harvested during elective surgical reconstruction and fixed for immunofluorescence. Green, HDAC4; blue, To-pro-3 (i.e. nuclear). D, human control (Con) and aortic aneurysm sections were immunostained for smooth muscle -actin and subjected to laser microdissection. Total RNA was isolated from collected cells and subjected to quantitative PCR for HDAC4 and GAPDH (n 4; *, p 0.05 was considered statistically significant).

Article Snippet: Cell Culture and Treatment of VSMCs—Rat A10 myoblasts (ATCC; CRL-1476) were maintained in growth medium consisting of 10% fetal bovine serum (FBS).

Techniques: Expressing, Transferring, Immunofluorescence, Control, Laser Capture Microdissection, Isolation, Real-time Polymerase Chain Reaction

Journal: Journal of Biological Chemistry

Article Title: Protein Kinase A-regulated Assembly of a MEF2·HDAC4 Repressor Complex Controls c-Jun Expression in Vascular Smooth Muscle Cells

doi: 10.1074/jbc.m109.000539

Figure Lengend Snippet: FIGURE 4. The MEF2 cis-element in the c-jun promoter acts as a repressor element in quiescent VSMCs. A,A10cellsweretransfectedwithawild-typec-junpromoter(pJC6),ac-junreporterwiththeMEF2bindingsite mutated (pJSX), or a c-jun reporter gene with the AP1 site mutated (pJTX). The cells were harvested for lucif- erase under growth conditions (i.e. 10% FBS) or in serum-free Dulbecco’s modified Eagle’s medium. B, A10 cells were transfected with wild-type c-Jun-luc or myocardin-luc, with MEF2A, HDAC4, or HDAC4 L175A, as indi- cated. C, VSMCs were transfected with a specific siRNA targeted to HDAC4 (siHDAC4) or a scrambled nonspe- cific oligonucleotide in pSilencer H3 (Ambion). Following transfection, positive cells were selected using puro- mycin, followed by immunoblot analysis. For luciferase, increasing amounts of siHDAC4 were transfected with wild-type c-Jun-luc. Growth arrested A10 cells were treated with trichostatin A (1 M, Sigma) for 2 h prior to harvesting. D, A10 cells were transfect siHDAC4 or scrambled control. Following recovery, positive cells were selectedusingpuromycin,followingbyovernightquiescenceinserum-freemedium.Thecellswerestimulated with 20 ng/ml of PDGF as indicated and subjected to immunoblot (IB) analysis (*, p 0.05 was considered statistically significant).

Article Snippet: Cell Culture and Treatment of VSMCs—Rat A10 myoblasts (ATCC; CRL-1476) were maintained in growth medium consisting of 10% fetal bovine serum (FBS).

Techniques: Modification, Transfection, Western Blot, Luciferase, Control

Journal: Journal of Biological Chemistry

Article Title: Protein Kinase A-regulated Assembly of a MEF2·HDAC4 Repressor Complex Controls c-Jun Expression in Vascular Smooth Muscle Cells

doi: 10.1074/jbc.m109.000539

Figure Lengend Snippet: FIGURE 6. PKA inhibits c-jun expression through HDAC4. A, growth phase VSMCs were treated with milri- none (10 M; Sigma) and isoproteronol (1 M) for 2 h followed by fixation with 4% paraformaldehyde. Fixed cells were then subjected to immunofluorescence (IF) with an HDAC4 primary antibody (Sigma). B, A10 cells wereserum-starvedandpretreatedwithforskolin(10M)andmilrinone(10M,Sigma),orMe2SO.Nuclearand cytosolic extractions were immunoblotted (IB) for HDAC4, MEF2D, and GAPDH. C, A10 cells were transfected with MEF2A and HDAC4 and treated for 2 h with isoproteronol and milrinone. Protein extracts were subjected to immunoprecipitation (IP) and immunoblotting as indicated. D, VSMCs were transfected with c-Jun-luc, siHDAC4, or PKA, as indicated (n.s., not significant).

Article Snippet: Cell Culture and Treatment of VSMCs—Rat A10 myoblasts (ATCC; CRL-1476) were maintained in growth medium consisting of 10% fetal bovine serum (FBS).

Techniques: Expressing, Immunofluorescence, Transfection, Immunoprecipitation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Protein Kinase A-regulated Assembly of a MEF2·HDAC4 Repressor Complex Controls c-Jun Expression in Vascular Smooth Muscle Cells

doi: 10.1074/jbc.m109.000539

Figure Lengend Snippet: FIGURE 7. PKA inhibits HDAC4 nuclear export through SIK1. A, VSMCs were transfected with HDAC4-GFP and SIK1 or empty pSVL. The following serum starvation micrographs were obtained by standard fluorescent techniques. Growth phase A10s in 10% FBS () or serum-free medium () were harvested for total RNA and subjected to reverse transcription-PCR for SIK1 and GAPDH. B and C, A10 cells were transfected with c-Jun-luc, SIK1, SIK1 S577A, or PKA, as indicated. D, COS7 cells were transfected with HDAC4-GFP and SIK1, SIK1 S577A, or PKA, as indicated. The micrographs were obtained following 24 h of recovery.

Article Snippet: Cell Culture and Treatment of VSMCs—Rat A10 myoblasts (ATCC; CRL-1476) were maintained in growth medium consisting of 10% fetal bovine serum (FBS).

Techniques: Transfection, Reverse Transcription

Journal: Journal of Biological Chemistry

Article Title: Protein Kinase A-regulated Assembly of a MEF2·HDAC4 Repressor Complex Controls c-Jun Expression in Vascular Smooth Muscle Cells

doi: 10.1074/jbc.m109.000539

Figure Lengend Snippet: FIGURE 8. Down-regulation of c-Jun is critical for VSMC differentiation. A, VSMCs were transfected with wild-type c-Jun-luc, c-Jun-luc containing a mutation in the MEF2 binding site (c-Jun mut), or a smooth muscle -actin reporter gene (SMA-luc). Quiescent cells were treated with 50 ng/ml of IGF-1 overnight and harvested for luciferase extracts. Protein extracts from overnight treated IGF-1 A10 cells were subjected to immunoblotting for c-Jun or SMA (Sigma). B, 10T1/2 fibroblasts were transfected with smooth myosin heavy chain (SM-MHC-luc), smooth muscle -actin (SMA-luc), or calponin (Calponin-luc) reporter genes with expression vectors for c-Jun and the smooth muscle isoform of myocardin (myocardin 856), as indicated. The cells were harvested for luciferase24hafterrecovery.C,10T1/2cellsweretransfectedwithmyocardin856andc-Jun,asindicated.Aftera24hrecovery,thecellswerere-fedin5%horse serum and allowed to differentiate for 4 days before harvesting for protein extracts and immunoblotting for SMA or SM-MHC (Biomedical Science). D, COS7 cells were transfected with c-Jun or myocardin 856, as indicated. Protein extracts were subjected to immunoprecipitation (IP) and immunoblotting (IB), as indicated. E, 10T1/2 cells were transfected with a Gal4 reporter gene, and Gal4-c-Jun fusion proteins containing full-length c-Jun (FL) or amino acids 1–67, with a myocardin-VP16 fusion protein, as indicated. The extracts were subject to luciferase assay. Con, control.

Article Snippet: Cell Culture and Treatment of VSMCs—Rat A10 myoblasts (ATCC; CRL-1476) were maintained in growth medium consisting of 10% fetal bovine serum (FBS).

Techniques: Transfection, Mutagenesis, Binding Assay, Luciferase, Western Blot, Expressing, Immunoprecipitation, Control

Journal: bioRxiv

Article Title: Anti-nucleolin aptamer, iSN04, inhibits the inflammatory responses in myoblasts by modulating the β-catenin/NF-κB signaling pathway

doi: 10.1101/2023.04.01.535227

Figure Lengend Snippet: iSN04 inhibits NF-κB-dependent inflammatory gene expression in myoblasts. (A) qPCR results of TNF-α ( Tnf ), IL-6 ( Il6 ), and NF-κB p65 subunit ( Rela ) expression in the C2C12 cells pre-treated with 10 μM iSN04 for 3 h and then treated with 50 ng/ml TNF-α, 100 ng/ml Pam 3 CSK 4 , or 100 ng/ml FSL-1 for 2 h. * p < 0.05, ** p < 0.01 vs control; † p < 0.05, †† p < 0.05 vs ligand. n = 3-4. (B) Relative NF-κB-Luc activities in the C2C12 cells pre-treated with 10 μM iSN04 for 3 h and then treated with 50 ng/ml TNF-α or 100 ng/ml Pam 3 CSK 4 for 40 h. ** p < 0.01 vs control; † p < 0.05, †† p < 0.05 vs ligand. n = 3.

Article Snippet: Murine myoblast cell line C2C12 (DS Pharma Biomedical, Osaka, Japan) [ , ], rat embryonic thoracic aorta smooth muscle cell line A10 (CRL-1476; ATCC, Manassas, VA, USA) [ , ], and murine 3T3-L1 fibroblast cell line (IFO50416; JCRB Cell Bank, Osaka, Japan) as a model of pre-adipocytes [ ] were cultured in DMEM (Nacalai, Osaka, Japan) supplemented with 10% fetal bovine serum (HyClone; GE Healthcare, Salt Lake City, UT, USA) and a mixture of 100 units/ml penicillin and 100 μg/ml streptomycin (Nacalai) at 37°C under 5% CO 2 throughout the experiments.

Techniques: Gene Expression, Expressing, Control

Journal: bioRxiv

Article Title: Anti-nucleolin aptamer, iSN04, inhibits the inflammatory responses in myoblasts by modulating the β-catenin/NF-κB signaling pathway

doi: 10.1101/2023.04.01.535227

Figure Lengend Snippet: iSN04 inhibits nuclear translocation of NF-κB. (A and B) Representative images of NF-κB staining of the C2C12 cells pre-treated with 10 μM iSN04 for 3 h and then treated with 50 ng/ml TNF-α (A) or 100 ng/ml Pam 3 CSK 4 (B) for 30 min. Scale bar, 50 μm.

Article Snippet: Murine myoblast cell line C2C12 (DS Pharma Biomedical, Osaka, Japan) [ , ], rat embryonic thoracic aorta smooth muscle cell line A10 (CRL-1476; ATCC, Manassas, VA, USA) [ , ], and murine 3T3-L1 fibroblast cell line (IFO50416; JCRB Cell Bank, Osaka, Japan) as a model of pre-adipocytes [ ] were cultured in DMEM (Nacalai, Osaka, Japan) supplemented with 10% fetal bovine serum (HyClone; GE Healthcare, Salt Lake City, UT, USA) and a mixture of 100 units/ml penicillin and 100 μg/ml streptomycin (Nacalai) at 37°C under 5% CO 2 throughout the experiments.

Techniques: Translocation Assay, Staining

Journal: bioRxiv

Article Title: Anti-nucleolin aptamer, iSN04, inhibits the inflammatory responses in myoblasts by modulating the β-catenin/NF-κB signaling pathway

doi: 10.1101/2023.04.01.535227

Figure Lengend Snippet: iSN04 inhibits GSK-3β phosphorylation and β-catenin activation. (A) Representative images of β-catenin staining of the C2C12 cells pre-treated with 10 μM iSN04 for 3 h and then treated with 50 ng/ml TNF-α for 1 h. Scale bar, 50 μm. (B) Representative images and quantification of Western blotting of β-catenin and GAPDH from C2C12 cells treated as in panel A . ** p < 0.01 vs control, † p < 0.05 vs TNF-α. n = 3.

Article Snippet: Murine myoblast cell line C2C12 (DS Pharma Biomedical, Osaka, Japan) [ , ], rat embryonic thoracic aorta smooth muscle cell line A10 (CRL-1476; ATCC, Manassas, VA, USA) [ , ], and murine 3T3-L1 fibroblast cell line (IFO50416; JCRB Cell Bank, Osaka, Japan) as a model of pre-adipocytes [ ] were cultured in DMEM (Nacalai, Osaka, Japan) supplemented with 10% fetal bovine serum (HyClone; GE Healthcare, Salt Lake City, UT, USA) and a mixture of 100 units/ml penicillin and 100 μg/ml streptomycin (Nacalai) at 37°C under 5% CO 2 throughout the experiments.

Techniques: Phospho-proteomics, Activation Assay, Staining, Western Blot, Control

Journal: Biomolecules

Article Title: Decapeptide from Potato Hydrolysate Induces Myogenic Differentiation and Ameliorates High Glucose-Associated Modulations in Protein Synthesis and Mitochondrial Biogenesis in C2C12 Cells

doi: 10.3390/biom12040565

Figure Lengend Snippet: DI-10 activates ERK, Akt, mTOR, and FOXO3a proteins and facilitates protein synthesis in myoblast cells. Dose-dependent activation of ERK, Akt, mTOR, and FOXO3a was observed in myoblast cells after DI-10. ( A ) Western blotting show phosphorylation of ERK, Akt, mTOR, and FOXO3A in C2C12 cells after DI-10 treatment. ( B ) Graph showing densitometric analysis. *** p < 0.001, and ** p < 0.01 compared to control group.

Article Snippet: C2C12 murine myoblast cells was purchased from Bioresource Collection and Research Center (BCRC, Taiwan) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) containing antibiotics streptomycin (100 μg/mL) and penicillin (100 U/mL) in a 10 cm culture plate.

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Control

Journal: Biomolecules

Article Title: Decapeptide from Potato Hydrolysate Induces Myogenic Differentiation and Ameliorates High Glucose-Associated Modulations in Protein Synthesis and Mitochondrial Biogenesis in C2C12 Cells

doi: 10.3390/biom12040565

Figure Lengend Snippet: DI-10 improves the viability of C2C12 myoblast cells under high glucose stress. ( A ) C2C12 cells were treated with different concentrations of D-glucose (5, 15, and 30 mM) and the cell viability was determined with MTT assay. ( B ) C2C12 cells were treated with 30 mM HG followed by different concentrations of DI-10. *** p < 0.001 compared to control groups and ### p < 0.001 compared to the high glucose.

Article Snippet: C2C12 murine myoblast cells was purchased from Bioresource Collection and Research Center (BCRC, Taiwan) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) containing antibiotics streptomycin (100 μg/mL) and penicillin (100 U/mL) in a 10 cm culture plate.

Techniques: MTT Assay, Control

Journal: Biomolecules

Article Title: Decapeptide from Potato Hydrolysate Induces Myogenic Differentiation and Ameliorates High Glucose-Associated Modulations in Protein Synthesis and Mitochondrial Biogenesis in C2C12 Cells

doi: 10.3390/biom12040565

Figure Lengend Snippet: Decapeptide DI-10 promotes myoblast differentiation under high-glucose conditions. Effect of decapeptide DI-10 on myogenic differentiation under hyperglycemic conditions was determined by ( A ) microscopic observation of cell morphology. Photomicrographs were obtained using an Olympus ® CKX53 microscope (100×) ( B ) MyHC expression in C2C12 cells under HG conditions. ### p < 0.001 compared to the high glucose.

Article Snippet: C2C12 murine myoblast cells was purchased from Bioresource Collection and Research Center (BCRC, Taiwan) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) containing antibiotics streptomycin (100 μg/mL) and penicillin (100 U/mL) in a 10 cm culture plate.

Techniques: Microscopy, Expressing

Journal: Biomolecules

Article Title: Decapeptide from Potato Hydrolysate Induces Myogenic Differentiation and Ameliorates High Glucose-Associated Modulations in Protein Synthesis and Mitochondrial Biogenesis in C2C12 Cells

doi: 10.3390/biom12040565

Figure Lengend Snippet: Effect of DI-10 on Akt, mTOR, and AMPK phosphorylation under hyperglycemic conditions in C2C12 cells. ( A ) Immuno blot showing phosphorylation of Akt, mTOR, and AMPK proteins involved in protein synthesis. ( B ) Graph showing densitometric analysis. ** p < 0.01 and * p < 0.05 compared to control group and # p < 0.05 and ### p < 0.001 compared to the high glucose.

Article Snippet: C2C12 murine myoblast cells was purchased from Bioresource Collection and Research Center (BCRC, Taiwan) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) containing antibiotics streptomycin (100 μg/mL) and penicillin (100 U/mL) in a 10 cm culture plate.

Techniques: Phospho-proteomics, Control

Journal: Biomolecules

Article Title: Decapeptide from Potato Hydrolysate Induces Myogenic Differentiation and Ameliorates High Glucose-Associated Modulations in Protein Synthesis and Mitochondrial Biogenesis in C2C12 Cells

doi: 10.3390/biom12040565

Figure Lengend Snippet: Bioactive peptides enhance myogenic differentiation and skeletal muscle protein synthesis under high-glucose conditions in C2C12 myoblast cells. ↑ increase, ↓ decrease.

Article Snippet: C2C12 murine myoblast cells was purchased from Bioresource Collection and Research Center (BCRC, Taiwan) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) containing antibiotics streptomycin (100 μg/mL) and penicillin (100 U/mL) in a 10 cm culture plate.

Techniques:

Journal: bioRxiv

Article Title: Anti-nucleolin aptamer, iSN04, inhibits the inflammatory responses in myoblasts by modulating the β-catenin/NF-κB signaling pathway

doi: 10.1101/2023.04.01.535227

Figure Lengend Snippet: iSN04 inhibits NF-κB-dependent inflammatory gene expression in VSMCs and pre-adipocytes. (A) qPCR results of IL-6 ( Il6 ), IL-8 ( Cxcl8 ), and MCP-1 ( Ccl2 ) expression in A10 cells pre-treated with 10 μM iSN04 for 3 h and subsequently treated with 50 ng/ml TNF-α for 4 h. * p < 0.05, ** p < 0.01 vs control; † p < 0.05, †† p < 0.05 vs TNF-α. n = 3. (B) Relative NF-κB-Luc activities in the A10 cells pre-treated with 10 μM iSN04 for 3 h and subsequently treated with 3 ng/ml TNF-α for 40 h. ** p < 0.01 vs control, † p < 0.05 vs TNF-α. n = 3. (C) qPCR results of TNF-α ( Tnf ) and IL-6 ( Il6 ) expression in the 3T3-L1 cells pre-treated with 30 μM iSN04 for 3 h and subsequently treated with 5 ng/ml TNF-α or 10 ng/ml Pam 3 CSK 4 for 2 h. * p < 0.05, ** p < 0.01 vs control; † p < 0.05, †† p < 0.05 vs ligand. n = 3-4.

Article Snippet: Murine myoblast cell line C2C12 (DS Pharma Biomedical, Osaka, Japan) [ , ], rat embryonic thoracic aorta smooth muscle cell line A10 (CRL-1476; ATCC, Manassas, VA, USA) [ , ], and murine 3T3-L1 fibroblast cell line (IFO50416; JCRB Cell Bank, Osaka, Japan) as a model of pre-adipocytes [ ] were cultured in DMEM (Nacalai, Osaka, Japan) supplemented with 10% fetal bovine serum (HyClone; GE Healthcare, Salt Lake City, UT, USA) and a mixture of 100 units/ml penicillin and 100 μg/ml streptomycin (Nacalai) at 37°C under 5% CO 2 throughout the experiments.

Techniques: Expressing