Journal: Mediators of Inflammation

Article Title: Elevation of IL-6 and IL-33 Levels in Serum Associated with Lung Fibrosis and Skeletal Muscle Wasting in a Bleomycin-Induced Lung Injury Mouse Model

doi: 10.1155/2019/7947596

Figure Lengend Snippet: IL-6 and IL-33 may synergistically cause muscle atrophy. (a) The quadriceps muscle of the mice was homogenized, and the lysate was analyzed by Western blotting with specific antibodies against STAT3, AMPK, and Atrogin-1. (b) C2C12 cells, mouse adherent myoblasts, were incubated with 2% horse serum for 72 hours and stimulated with recombinant mouse IL-6 and IL-33 in serum-free medium as indicated for 24 hours. The remaining cells were harvested, and the levels of p-STAT3, STAT3, p-AMPK α , and AMPK α in the cell lysate were analyzed by Western blotting. α -Tubulin and GAPDH were used as internal controls. The intensity of bands in the Western blots was measured by ImageJ software. The quantitative data were expressed as the means ± SD. ∗ p < 0.05 compared with control.

Article Snippet: C2C12 mouse adherent myoblasts were obtained from BCRC (Bioresource Collection and Research Center, Hsinchu, Taiwan).

Techniques: Western Blot, Incubation, Recombinant, Software, Control

Journal: International Journal of Molecular Sciences

Article Title: IRE1-XBP1 Pathway of the Unfolded Protein Response Is Required during Early Differentiation of C2C12 Myoblasts

doi: 10.3390/ijms21010182

Figure Lengend Snippet: IRE1 is required in C2C12 differentiation. ( a ) IRE1-knockdown cell lines #1, #2, and mock cells were evaluated for the expression of IRE1/ Ern1 mRNA. Results are means + SEM (three biological replicates). ** p < 0.01. ( b ) Differentiation was induced in IRE1-knockdown cell lines and mock cells. After 48 h, cells were observed under phase contrast. Black arrows show the immature myotubes. Scale bar = 100 µm. ( c , d ) Cells were harvested after differentiation induction at 48 h. mRNA expression of myogenic factors ( c ) and UPR relative factors ( d ) was analyzed by qPCR. Results are means + SEM (three biological replicates). * p < 0.05, ** p < 0.01.

Article Snippet: C2C12 mouse myoblasts were obtained from DS Pharma Biomedical (Osaka, Japan).

Techniques: Knockdown, Expressing

Journal: International Journal of Molecular Sciences

Article Title: IRE1-XBP1 Pathway of the Unfolded Protein Response Is Required during Early Differentiation of C2C12 Myoblasts

doi: 10.3390/ijms21010182

Figure Lengend Snippet: IRE1 ribonuclease activity is required in early phase of C2C12 differentiation. ( a ) Differentiation was induced in the presence or absence of IRE1 RNase inhibitor, STF-083010 (60 µM; black bars) or DMSO (gray bars) for various time intervals as indicated. ( b ) Identification of critical time period for inhibitory effect of IRE1 activity on C2C12 differentiation. Scale bar = 200 µm. ( c ) Fusion index of STF-083010- or DMSO-treated cells. Results are mean + SEM (three biological replicates). The different letters denote significant differences between groups at p < 0.05 by Tukey’s HSD test.

Article Snippet: C2C12 mouse myoblasts were obtained from DS Pharma Biomedical (Osaka, Japan).

Techniques: Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: IRE1-XBP1 Pathway of the Unfolded Protein Response Is Required during Early Differentiation of C2C12 Myoblasts

doi: 10.3390/ijms21010182

Figure Lengend Snippet: XBP1 is required for C2C12 differentiation. ( a ) mRNA expression of Xbp1 and spliced Xbp1 were compared between XBP1-knockdown cells (XBP1-KD) and mock cells. Results are means + SEM (three biological replicates). Student’s t -test. ** p < 0.01. ( b ) XBP1-knockdown cells and mock cells were induced to differentiate until day 5. Cells were observed for immunofluorescent staining with anti-MHC antibody. Scale bar = 200 µm. ( c ) Fusion index of mock or XBP1-KD cells. Results are mean + SEM (three biological replicates). Student’s t -test. ** p < 0.01. ( d ) Cells were harvested on the indicated day. mRNA expression of each myogenic factor was analyzed by qPCR. Results are means + SEM (three biological replicates). Student’s t -test. * p < 0.05, ** p < 0.01.

Article Snippet: C2C12 mouse myoblasts were obtained from DS Pharma Biomedical (Osaka, Japan).

Techniques: Expressing, Knockdown, Staining

Journal: International Journal of Molecular Sciences

Article Title: IRE1-XBP1 Pathway of the Unfolded Protein Response Is Required during Early Differentiation of C2C12 Myoblasts

doi: 10.3390/ijms21010182

Figure Lengend Snippet: CDK5 is a downstream target of IRE1-XBP1 in C2C12 cells. ( a ) mRNA expression of Xbp1 and Cdk5 during C2C12 differentiation. Results are means + SEM (three biological replicates). Student’s t -test. ** p < 0.01. ( b ) An illustration of the predicted mouse Cdk5 promoter region. The region upstream of the Cdk5 gene comprises three XBP1-binding domains including CCAAT at −544 bp, TGCCACGTGG at −597 bp, and CCACGT at −1112 bp from the transcription start site. ( c , d ) Chromatin immunoprecipitation assay using a C2C12 genomic sample. Input DNA = positive control. Rabbit IgG was used as negative control ChIP ( c ). ChIP assay was performed by quantitative PCR analysis ( d ). Results are means + SEM (three biological replicates). Student’s t -test. * p < 0.05. ( e ) XBP1-knockdown and mock cells were transfected with the vector containing the Cdk5 promoter construct ( p 1400) or an empty vector. Cdk5 promoter activity was assessed by luciferase assay. Results are means + SEM (three biological replicates). Student’s t -test. ** p < 0.01.

Article Snippet: C2C12 mouse myoblasts were obtained from DS Pharma Biomedical (Osaka, Japan).

Techniques: Expressing, Binding Assay, Chromatin Immunoprecipitation, Positive Control, Negative Control, Real-time Polymerase Chain Reaction, Knockdown, Transfection, Plasmid Preparation, Construct, Activity Assay, Luciferase

Journal: Skeletal Muscle

Article Title: MiR-1290 promotes myoblast differentiation and protects against myotube atrophy via Akt/p70/FoxO3 pathway regulation

doi: 10.1186/s13395-021-00262-9

Figure Lengend Snippet: The effect of miR-1290 mimic transfection on C2C12 cells. a and b The MHC staining of C2C12 myoblast with miR-1290 or miR-NC and quantification of MHC area (scale bar, 50 μm). c – f The western blot analysis and quantification of MHC, MyoD, and MyoG after transfection of miRNAs. GAPDH was used as an internal control for western blot analysis. The statistical difference among miR-1290 transfection group and control group were considered significant at the levels of * P < 0.05, ** P < 0.01, or *** P < 0.001

Article Snippet: The mouse myoblast C2C12 cells were provided by Cyagen Biosciences, Inc., and cultured in DMEM containing 1% penicillin-streptomycin and 10% fetal bovine serum following the American Type Culture collection instructions.

Techniques: Transfection, Staining, Western Blot, Control

Journal: Skeletal Muscle

Article Title: MiR-1290 promotes myoblast differentiation and protects against myotube atrophy via Akt/p70/FoxO3 pathway regulation

doi: 10.1186/s13395-021-00262-9

Figure Lengend Snippet: The effect of miR-1290 mimic transfection on C2C12 cells under TNF-α. a and b Giemsa staining and myotube diameters among all groups. c – e The western blot analysis and quantification of MuRF1 and atrogin-1 after overexpression of miR-1290 in TNF-α-induced atrophy. GAPDH was used as an internal control for western blot analysis. The statistical difference among miR-1290 transfection group and other groups were considered significant at the levels of * P < 0.05, ** P < 0.01, or *** P < 0.001

Article Snippet: The mouse myoblast C2C12 cells were provided by Cyagen Biosciences, Inc., and cultured in DMEM containing 1% penicillin-streptomycin and 10% fetal bovine serum following the American Type Culture collection instructions.

Techniques: Transfection, Staining, Western Blot, Over Expression, Control

Journal: Skeletal Muscle

Article Title: MiR-1290 promotes myoblast differentiation and protects against myotube atrophy via Akt/p70/FoxO3 pathway regulation

doi: 10.1186/s13395-021-00262-9

Figure Lengend Snippet: The intrinsic mechanism of miR-1290’s effect. a Bioinformatics analysis was performed to predict the miR-1290-binding seed sequence in the 3’UTR of FoxO3. b The luciferase result of miR-1290 and FOXO3. c and d The western blot analysis and quantification to determine FoxO3 levels in cytoplasm and nucleus in miR-1290-transfected C2C12 myoblast. e and f The knockdown efficiency of FoxO3-specific siRNA was confirmed by western blot. g and h MHC staining was performed after FoxO3 knockdown (scale bar, 50 μm). i and j . The expression of MyoD and MyoG were analyzed by western blot. GAPDH and Lamin B1 are cytoplasmic and nuclear protein loading controls, respectively. The statistical difference among miR-1290 transfection group and other groups were considered significant at the levels of * P < 0.05, ** P < 0.01, or *** P < 0.001

Article Snippet: The mouse myoblast C2C12 cells were provided by Cyagen Biosciences, Inc., and cultured in DMEM containing 1% penicillin-streptomycin and 10% fetal bovine serum following the American Type Culture collection instructions.

Techniques: Binding Assay, Sequencing, Luciferase, Western Blot, Transfection, Knockdown, Staining, Expressing

Journal: Skeletal Muscle

Article Title: MiR-1290 promotes myoblast differentiation and protects against myotube atrophy via Akt/p70/FoxO3 pathway regulation

doi: 10.1186/s13395-021-00262-9

Figure Lengend Snippet: MiR-1290 activates AKT/P70/FoxO3 signaling pathways during myoblast differentiation. a MHC staining performed after treated miR-1290/miR-NC with or without GDC-0068 (scale bar, 50 μm). b MHC-positive areas/total areas were quantified using Harmony 4.1 software ( n = 6). c – f The western blot analysis and quantification of phosphorylated and all forms of AKT and P70, MyoG, and MyoD, after transfecting miR-1290/miR-NC with or without GDC0068. GDC-0068 inhibited miR-1290-activated phosphorylation of AKT and P70 in C2C12 myoblasts. Western blot to analyze FoxO3 expression levels in cytoplasm and nucleus of C2C12 myoblasts. GAPDH and Lamin B1 are cytoplasmic and nuclear protein loading controls, respectively. The statistical difference among miR-1290 transfection group and inhibitor group were considered significant at the levels of * P < 0.05, ** P < 0.01, or *** P < 0.001

Article Snippet: The mouse myoblast C2C12 cells were provided by Cyagen Biosciences, Inc., and cultured in DMEM containing 1% penicillin-streptomycin and 10% fetal bovine serum following the American Type Culture collection instructions.

Techniques: Protein-Protein interactions, Staining, Software, Western Blot, Phospho-proteomics, Expressing, Transfection

Journal: Skeletal Muscle

Article Title: MiR-1290 promotes myoblast differentiation and protects against myotube atrophy via Akt/p70/FoxO3 pathway regulation

doi: 10.1186/s13395-021-00262-9

Figure Lengend Snippet: Role of Protein kinase B (AKT)/P70/FOXO3 signaling pathway in effect of miR-1290 on myotube atrophy. a Giemsa staining was performed to calculate myotube diameters for TNF-α + miR-NC or TNF-α + miR-NC + GDC0068, TNF-α + miR-1290 or TNF-α + miR-1290 + GDC0068 treatments. b Cell diameters of five groups were measured. c – f Western blot analysis and quantification of phosphorylated and all forms of AKT and P70 of C1C12 myotubes for TNF-α + miR-NC or TNF-α + miR-NC + GDC0068, TNF-α + miR-1290 or TNF-α + miR-1290 + GDC0068 treatments. Western blot was performed to analyze the expression of MuRF1 and atrogin-1 in TNF-α + miR-NC or TNF-α + miR-NC + GDC0068, TNF-α + miR-1290 or TNF-α + miR-1290 + GDC0068 groups. After treatment with miR-1290/miR-NC or with GDC-0068, FoxO3 expression levels in cytoplasm and nucleus of C2C12 myotubes were examined by western blot. GAPDH and Lamin B1 are cytoplasmic and nuclear protein loading controls, respectively. The statistical difference among miR-1290 transfection group and inhibitor group were considered significant at the levels of * P < 0.05, ** P < 0.01, or *** P < 0.001

Article Snippet: The mouse myoblast C2C12 cells were provided by Cyagen Biosciences, Inc., and cultured in DMEM containing 1% penicillin-streptomycin and 10% fetal bovine serum following the American Type Culture collection instructions.

Techniques: Staining, Western Blot, Expressing, Transfection