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: International Journal of Cell Biology

Article Title: Interaction of Wnt Signaling with BMP/Smad Signaling during the Transition from Cell Proliferation to Myogenic Differentiation in Mouse Myoblast-Derived Cells

doi: 10.1155/2013/616294

Figure Lengend Snippet: Myosin heavy chain (MyHC) expression in parental C2C12 cells and C2C12-derived cells permanently expressing Wnt4. Parental C2C12 cells (a, b, e, and f) and W4-08 cells (c, d, g, and h) were cultured for 2 days in proliferation medium containing 10% fetal bovine serum (a–d), or in differentiation medium containing 2% horse serum (e–h), and then immunohistochemically stained with anti-MyHC antibodies, followed by counterstaining with DAPI. Spontaneous expression of slow-type MyHC was evident in W4-08 cells in proliferation medium and intensified in differentiation medium, although the proliferation rates were greatly reduced compared to those of the parental C2C12 cells, as observed in the reduced number of nuclei (blue).

Article Snippet: The C2C12 cell line (myoblast-like cell line from the C3H mouse) was purchased from the RIKEN Cell Bank (RIKEN, Wako, Japan) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Nichirei Corp., Tokyo).

Techniques: Expressing, Derivative Assay, Cell Culture, Staining

Journal: International Journal of Cell Biology

Article Title: Interaction of Wnt Signaling with BMP/Smad Signaling during the Transition from Cell Proliferation to Myogenic Differentiation in Mouse Myoblast-Derived Cells

doi: 10.1155/2013/616294

Figure Lengend Snippet: Summary of expression array analysis for C2C12 differentiation and Wnt4 expression. Red and green colors show upregulated and downregulated expression, respectively, with differentiation medium and Wnt4 overexpression. 1477 and 1836 genes were upregulated and downregulated, respectively, in differentiation medium and Wnt4 overexpression at a twofold magnitude. Refer to original data in supplementary 1 for details.

Article Snippet: The C2C12 cell line (myoblast-like cell line from the C3H mouse) was purchased from the RIKEN Cell Bank (RIKEN, Wako, Japan) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Nichirei Corp., Tokyo).

Techniques: Expressing, Over Expression

Journal: International Journal of Cell Biology

Article Title: Interaction of Wnt Signaling with BMP/Smad Signaling during the Transition from Cell Proliferation to Myogenic Differentiation in Mouse Myoblast-Derived Cells

doi: 10.1155/2013/616294

Figure Lengend Snippet: Effect of BMP4 and noggin on the myogenic differentiation of C2C12 cells. (a–d) The recombinant proteins were added to the proliferation medium at final concentrations of 5 ng/mL BMP4 and/or 50 ng/mL noggin and cultured in proliferation medium for 3 days, followed by immunostaining with anti-troponin T antibodies (red) and anti-phosphohistone H3 (Ser10) antibodies (green). BMP4 addition inhibited myogenic differentiation in proliferation medium. The number of cells expressing phosphorylated histone H3 was increased by adding noggin but not BMP4. (a) Control; (b) noggin; (c) BMP4; (d) noggin + BMP4. (e–g) The ratios of phosphohistone H3 (e) and troponin T-positive cells (f) to the total cell number were estimated. The number of multinuclear cells expressing troponin T (g) was also estimated to determine the ratio of fused cells among the total cells. Mean + SD ( n = 3), * P < 0.05 versus control with Student's t -test.

Article Snippet: The C2C12 cell line (myoblast-like cell line from the C3H mouse) was purchased from the RIKEN Cell Bank (RIKEN, Wako, Japan) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Nichirei Corp., Tokyo).

Techniques: Recombinant, Cell Culture, Immunostaining, Expressing

Journal: International Journal of Cell Biology

Article Title: Interaction of Wnt Signaling with BMP/Smad Signaling during the Transition from Cell Proliferation to Myogenic Differentiation in Mouse Myoblast-Derived Cells

doi: 10.1155/2013/616294

Figure Lengend Snippet: Effects of BMP4 and noggin on phospho-Smad1/5 expression with or without Wnt3a and Wnt4. (a–l) Recombinant adenoviruses were used to express Wnt3a , Wnt4 , or eGFP (control) uniformly in C2C12 cells. Cells were infected 48 hours earlier with an MOI of 400, and the recombinant proteins were added as shown in with final concentrations of 5 ng/mL BMP4 and/or 250 ng/mL noggin. Two hours after BMP4 addition, immunostaining for phospho-Smad1/5 was carried out for counting. (a–c) Control; (d–f) BMP4; (g–i) noggin; (j–l) BMP4 + noggin. (m) The ratio of the nuclear phospho-Smad1/5-positive cells to the total cell number was estimated in three independent experiments counted for 6 fields each (mean + SD, n = 18, * P < 0.05 with Student's t -test).

Article Snippet: The C2C12 cell line (myoblast-like cell line from the C3H mouse) was purchased from the RIKEN Cell Bank (RIKEN, Wako, Japan) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Nichirei Corp., Tokyo).

Techniques: Expressing, Recombinant, Infection, Immunostaining

Journal: International Journal of Cell Biology

Article Title: Interaction of Wnt Signaling with BMP/Smad Signaling during the Transition from Cell Proliferation to Myogenic Differentiation in Mouse Myoblast-Derived Cells

doi: 10.1155/2013/616294

Figure Lengend Snippet: Effects of BMP4 and noggin on β-catenin localization with or without Wnt3a and Wnt4. (a–i) Recombinant adenoviruses were used to express Wnt3a , Wnt4 , or eGFP (control) uniformly in C2C12 cells. Cells were infected 48 hours earlier with an MOI of 400, and the recombinant proteins were added as shown in with final concentrations of 5 ng/mL BMP4 and/or 250 ng/mL noggin. Two hours after BMP4 addition, immunostaining for β-catenin was carried out for counting. (j) Comparison of the β-catenin signals after adenovirus-mediated expression of Wnt3a and eGFP , after pixel intensity analysis for immunohistochemical signals with BZ-II Analyzer, shown in abscissa for signal intensity and ordinate for frequency. Wnt3a intensified signals to a higher level. (k and l) Comparison of the β-catenin signals after adenovirus-mediated expression of Wnt3a , Wnt4, and eGFP , in the presence or absence of BMP4 and/or noggin. Immunostaining for β-catenin was analyzed by digitizing the signals with BZ-II Analyzer for comparison. Typical results are shown. (k) Without BMP4; (l) with BMP4.

Article Snippet: The C2C12 cell line (myoblast-like cell line from the C3H mouse) was purchased from the RIKEN Cell Bank (RIKEN, Wako, Japan) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Nichirei Corp., Tokyo).

Techniques: Recombinant, Infection, Immunostaining, Expressing, Immunohistochemical staining

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

Journal: Translational Cancer Research

Article Title: Regulation of dynamin-related protein 1 (DRP1) levels modulates myoblast atrophy induced by C26 colon cancer-conditioned medium

doi: 10.21037/tcr-21-751

Figure Lengend Snippet: C26 CM induced myotube atrophy and activated DRP1 expression. (A) C2C12 myotubes visualized by a biologic microscopy at ×200 magnification and. (B) Analysis of myotubes’ mean diameters. (C) DRP1 protein levels of the HS/CM group by western blotting. Data were shown as mean ± SD. ***P<0.01. HS, myotube cultured in DMEM of 2% HS (horse serum); CM, myotube cultured in DMEM of 33% C26 CM (conditional medium); DRP1, dynamin-related protein 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: C26 BALB/c mouse colon adenocarcinoma cells and C2C12 C3H mouse myoblasts (CRL-2638 and CRL1772, respectively; American Type Culture Collection, Manassas, VA, USA) were obtained from Dr. Han (Zhongshan Hospital, Shanghai, China).

Techniques: Expressing, Microscopy, Western Blot, Cell Culture

Journal: Translational Cancer Research

Article Title: Regulation of dynamin-related protein 1 (DRP1) levels modulates myoblast atrophy induced by C26 colon cancer-conditioned medium

doi: 10.21037/tcr-21-751

Figure Lengend Snippet: Regulation of DRP1 levels modulates myotube wasting during C26 CM-induced muscle atrophy. (A) Fluorescence images of eGFP expression and. (B) DRP1 protein levels of KD/OE/Dc group. (C) C2C12 myotubes visualized by a biologic microscopy and. (D) Analysis of myotubes’ mean diameters of KD/OE/Dc group. Data were shown as mean ± SD. ***P<0.01 vs. control. eGFP, enhanced green fluorescent protein. HS, myotube cultured in DMEM of 2% HS (horse serum); CM, myotube cultured in DMEM of 33% C26 CM (conditional medium); Dc, myotube infected with control lentivirus in CM; OE, myotube infected with overexpressed lentivirus in CM; KD, myotube infected with knockdown lentivirus in CM; DRP1, dynamin-related protein 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: C26 BALB/c mouse colon adenocarcinoma cells and C2C12 C3H mouse myoblasts (CRL-2638 and CRL1772, respectively; American Type Culture Collection, Manassas, VA, USA) were obtained from Dr. Han (Zhongshan Hospital, Shanghai, China).

Techniques: Fluorescence, Expressing, Microscopy, Control, Cell Culture, Infection, Knockdown

Journal: Translational Cancer Research

Article Title: Regulation of dynamin-related protein 1 (DRP1) levels modulates myoblast atrophy induced by C26 colon cancer-conditioned medium

doi: 10.21037/tcr-21-751

Figure Lengend Snippet: Transcriptome analysis of C2C12 myotubes. (A) MA plot (M-values: log-intensity ratios/A-values: log-intensity averages) based on the mean value of gene expression and the fold change, red represents up-regulated genes and blue represents down-regulated genes. (B) Heatmap for clustering analysis of differential expressed genes, blue represents high-expressed genes and red represents low-expressed genes. (C) Scatter plot of enrichment KEGG pathways based on differential expressed genes, the most prominent 30 pathways were selected. Q-values are numbers transferred from adjusted Q-values by hypergeometric test. The green degree of enrichment represents the ratio: the differential expressed genes to the total annotation genes in this pathway. (D&E) Enrichment of KEGG pathway for ribosome and oxidative phosphorylation, red represents up-regulated genes, blue represents down-regulated genes. CM, myotube cultured in DMEM of 33% C26 conditional medium; Dc, myotube infected with control lentivirus in C26 conditional medium; KD, myotube infected with knockdown lentivirus in C26 conditional medium.

Article Snippet: C26 BALB/c mouse colon adenocarcinoma cells and C2C12 C3H mouse myoblasts (CRL-2638 and CRL1772, respectively; American Type Culture Collection, Manassas, VA, USA) were obtained from Dr. Han (Zhongshan Hospital, Shanghai, China).

Techniques: Gene Expression, Phospho-proteomics, Cell Culture, Infection, Control, Knockdown

Journal: Translational Cancer Research

Article Title: Regulation of dynamin-related protein 1 (DRP1) levels modulates myoblast atrophy induced by C26 colon cancer-conditioned medium

doi: 10.21037/tcr-21-751

Figure Lengend Snippet: C26 CM reduced the marker of ribosomal biogenesis at mRNA levels. Gene expression levels of Pol-I, c-Myc, Ubf, Nop56, Ncl from C2C12 myotubes in KD/Dc/CM/HS groups using a real-time PCR method (n=4). Data were shown as mean ± SD. *P<0.05. HS, myotube cultured in DMEM of 2% HS (horse serum); CM, myotube cultured in DMEM of 33% C26 conditional medium; Dc, myotube infected with control lentivirus in C26 conditional medium; KD, myotube infected with knockdown lentivirus in C26 conditional medium; Pol-I, RNA polymerase I; c-Myc, MYC proto-oncogene; Ubf, upstream binding factor; Nop56, nucleolar protein 56; Ncl, nucleolin; PCR, polymerase chain reaction.

Article Snippet: C26 BALB/c mouse colon adenocarcinoma cells and C2C12 C3H mouse myoblasts (CRL-2638 and CRL1772, respectively; American Type Culture Collection, Manassas, VA, USA) were obtained from Dr. Han (Zhongshan Hospital, Shanghai, China).

Techniques: Marker, Gene Expression, Real-time Polymerase Chain Reaction, Cell Culture, Infection, Control, Knockdown, Binding Assay, Polymerase Chain Reaction

Journal: Cell Communication and Signaling : CCS

Article Title: miR-449a regulates insulin signalling by targeting the Notch ligand, Jag1 in skeletal muscle cells

doi: 10.1186/s12964-019-0394-7

Figure Lengend Snippet: miR-449a targets Jag1 by binding to its 3’UTR. a Differentiated C2C12 cells were transfected with either the scramble (Scr) or the miR-449a mimic (1–75 nM). On termination of incubation (48 h), cells were lysed and 40 μg protein was resolved on SDS-PAGE and subjected to Western blot analysis using anti-Jag1 antibody. Hsc70 was taken as the loading control. b Differentiated C2C12 cells were incubated with miR-449a mimic (10 nM) alone or together with its inhibitor (10 nM). Control cells were transfected with scramble (Scr). After 48 h, Jag1 protein levels were assessed by Western blot analysis and Hsc70 was taken as the loading control. c C2C12 cells incubated as in ( b ) were assessed for the transcript levels of Jag1 by qRT-PCR. 18S rRNA was used as the normalisation control. d C2C12 cells were transfected with either scramble (Scr) or the miR-449a inhibitor (10, 25 nM) and incubated for 48 h. Protein levels of Jag1 were evaluated by western blot analysis and Hsc70 was taken as loading control. e Human primary skeletal muscle cells were transfected with either the scramble (Scr) or the miR-449a mimic (10 nM) alone or with its inhibitor and western blot analysis was performed using anti-Jag1 antibody. HSC70 was used as the loading control. f C2C12 cells were plated in 12-well plates and transfected with the wild-type (WT) or the mutated (mut) Jag1 3′ UTR (100 ng) together with the miR-449a mimic (10 nM) and/or its inhibitor (10 nM). Control cells were transfected with the scramble (Scr). After 48 h, cells were lysed and luciferase activity was measured as described in the ‘Materials and methods’. Firefly luciferase values were normalised to the values of Renilla luciferase. g The protein expression of Jag1 was assessed in db/+ and db/db mice by western blot analysis. Briefly, 40 μg protein from skeletal muscle of db/+ and db/db mice were separated on SDS-PAGE, transferred to nitro-cellulose membranes and probed with anti-Jag1 antibody. Hsc70 was used as the loading control. Densitometric analysis is given along with the respective blots. All experiments were done at least thrice and values present are mean ± SEM. *** p < 0.001, ** p < 0.01, * p < 0.05

Article Snippet: C2C12 mouse myoblast cells were obtained from the National Centre for Cell Science (NCCS), Pune, India and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal calf serum (Life Technologies, CA, USA) along with 100 units/ml penicillin, 0.1 mg/ml streptomycin (GIBCO, NY, USA) in the presence of 1.5 g/L sodium bicarbonate at 37 °C and 5% CO 2 .

Techniques: Binding Assay, Transfection, Incubation, SDS Page, Western Blot, Control, Quantitative RT-PCR, Luciferase, Activity Assay, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: miR-449a regulates insulin signalling by targeting the Notch ligand, Jag1 in skeletal muscle cells

doi: 10.1186/s12964-019-0394-7

Figure Lengend Snippet: miR-449a binding to Jag1 inhibits Notch target genes and NICD levels. a Differentiated C2C12 cells were transfected with the miR-449a mimic (10 nM) in the absence or presence of its inhibitor (10 nM). After 48 h, cells were lysed and relative transcript levels of Notch target genes, Hes1 ( a ) and Hey1 ( b ) were quantified by qRT-PCR and normalised to 18S rRNA. Control cells were transfected with the scramble (Scr). c Differentiated C2C12 cells were grown on six-well plates and transfected with miR-449a (10 nM) alone or in the presence of its inhibitor (10 nM). After 48 h, cells were fixed in 4% formaldehyde and immunofluorescence was performed with anti-NICD antibody as described in ‘Materials and Methods’ ( d ) Human primary skeletal muscle cells were grown on chamber slides and transfected with either scramble (Scr) or miR-449a alone or with its inhibitor (10 nM). As in (c), immunofluorescence was performed and cells were visualised in fluorescence microscope. Incubations were repeated at least three times and values are presented as mean ± SEM. *** p < 0.001, * p < 0.05

Article Snippet: C2C12 mouse myoblast cells were obtained from the National Centre for Cell Science (NCCS), Pune, India and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal calf serum (Life Technologies, CA, USA) along with 100 units/ml penicillin, 0.1 mg/ml streptomycin (GIBCO, NY, USA) in the presence of 1.5 g/L sodium bicarbonate at 37 °C and 5% CO 2 .

Techniques: Binding Assay, Transfection, Quantitative RT-PCR, Control, Immunofluorescence, Fluorescence, Microscopy

Journal: Cell Communication and Signaling : CCS

Article Title: miR-449a regulates insulin signalling by targeting the Notch ligand, Jag1 in skeletal muscle cells

doi: 10.1186/s12964-019-0394-7

Figure Lengend Snippet: Overexpression of miR-449a and inhibition of Notch signalling promote insulin signalling. a Differentiated C2C12 cells were transfected with the miR-449a mimic (10 nM) alone or together with its inhibitor (10 nM). Control cells were transfected with the scramble (Scr). After 48 h, cells of all groups were treated with insulin (100 nM) for 20 min and on termination of incubation, they were lysed and probed for the levels of p-PI3K, PI3K ( a ), p-AKT, AKT ( b ) by western blot analysis. β-actin was used as loading control. c C2C12 cells were transfected with either the scramble or miR-449a alone or together with its inhibitor and after 48 h, were incubated in Kreb’s-Ringer bicarbonate buffer for 2 h. Cell were then pre-incubated with insulin (100 nM) for 15 min, followed by incubation with 2-NBDG (500 μM) in the presence of insulin (100 nM) for 1 h. On termination of incubation, fluorescence intensity of 2-NBDG was measured as described in ‘Materials and Methods’ section. d Differentiated C2C12 cells were incubated with the Notch inhibitor, DAPT (5,10 μM) for 24 h and the transcript levels of Notch target gene, Hes1 were quantified by qRT-PCR. Control cells were incubated in the presence of DMSO (C). 18S rRNA was used as loading control. In another experiment, cells were incubated as in ( d ) for 24 h and then incubated with insulin (100 nM) for 20 min and evaluated for the levels of p-PI3K, PI3K ( e ) and p-AKT,AKT ( f ). β-actin was used as the loading control. Densitometric analyses are given below the respective blots. All the experiments were done at least thrice and data presented as mean ± SEM. * p < 0.05, ** p < 0.01

Article Snippet: C2C12 mouse myoblast cells were obtained from the National Centre for Cell Science (NCCS), Pune, India and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal calf serum (Life Technologies, CA, USA) along with 100 units/ml penicillin, 0.1 mg/ml streptomycin (GIBCO, NY, USA) in the presence of 1.5 g/L sodium bicarbonate at 37 °C and 5% CO 2 .

Techniques: Over Expression, Inhibition, Transfection, Control, Incubation, Western Blot, Fluorescence, Quantitative RT-PCR

Journal: Nature Communications

Article Title: MyoD1 localization at the nuclear periphery is mediated by association of WFS1 with active enhancers

doi: 10.1038/s41467-025-57758-x

Figure Lengend Snippet: a Schematic overview of experimental strategy for monitoring the 3D nuclear position of the MyoD1 locus in living myoblasts by inserting a 64x LacO array downstream of MyoD1 coupled with expression of GFP-LacR. b Representative 3D reconstructed z stack of images showing intranuclear 3D position of the MyoD1 locus in proliferating myoblasts (green dots). Scale bars are indicated on images. See also Supplementary Movie . c Immunofluorescence microscopy of proliferating and 4-days differentiated MyoD1 and Pax7 reporter myoblasts. MyoD1 and Pax7 loci are indicated by yellow arrowheads. Lamin A/C staining (red) marks nuclear border. Scale bars, 10 μm. d Schematic drawing depicting the analysis of the radial distance of gene loci to the nuclear periphery and normalization based on nuclear height. e Density plots of the normalized radial distance of loci to the periphery. Left: Distribution of MyoD1 in proliferating (red) and differentiating (day 4, green) C2C12 myoblasts. n prolif = 106, n diff = 120, p = 1 × 10 −4 . Right: Distribution of MyoD1 and Pax7 loci in proliferating myoblasts. n MyoD1 = 208, n Pax7 = 79, p = 6.3 × 10 −4 . p values for loci were calculated using two-sample, two-sided KS test and corrected for multiple testing using Hochberg method. Numeric values displayed on violin plots represent median values. f Normalized radial position distribution of data shown in ( e ) displayed in violin plots. H3K9me2-marked region (gray-shaded horizontal bar) represents peripheral heterochromatin layer as measured in ( g ). **** p = 1 × 10 − 4 , *** p = 6.3 × 10 −4 p values were calculated using two-sample, two-tailed KS test and corrected for multiple testing using Hochberg method. g H3K9me2 immunofluorescence staining of MyoD1 C2C12 reporter myoblasts. Width of H3K9me2-marked layer is measured in rendered images (middle panel, yellow dots and lines illustrate measurement method for heterochromatin layer), bar graph depicts normalized mean thickness ± SEM of heterochromatin layer measured at 100 different locations. Scale bars, 10 μm. Images in ( c ) were processed with Fiji and in ( b ) and ( g ) with Imaris. Source data are provided as a file.

Article Snippet: To generate MyoD1 and Pax7 reporter cell lines, C2C12 mouse myoblasts were transfected with the vector pSpCas9(BB)-2A-GFP (pX458, plasmid #48138, Addgene) containing an sgRNA targeting a region 1 kb downstream of MyoD1 or Pax7 , as well as a repair template construct carrying the 64xLacO repeats flanked by regions homologous to MyoD1 and Pax7 , respectively (see also , chapter “Generation of repair template vector” and Supplementary Table for primer sequences).

Techniques: Expressing, Immunofluorescence, Microscopy, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: MyoD1 localization at the nuclear periphery is mediated by association of WFS1 with active enhancers

doi: 10.1038/s41467-025-57758-x

Figure Lengend Snippet: a Immunoblot analysis of total cell extracts of proliferating wildtype and knockout C2C12 clones using antibodies to the indicated antigens. Normalized radial distance of MyoD1 to the nuclear border in wildtype C2C12 myoblasts versus cells with ( b ) single knockouts, and ( c ) double or triple knockouts of indicated proteins. H3K9me2-marked heterochromatin layer is shown as gray-shaded bar. Dotted line indicates that experiments were performed separately using slightly different laser settings. n Wt = 208, n emerin KO = 333, n LAP2β KO = 140; n Wt = 141, n LA/C KO = 403, n LBR KO = 283; n Wt = 722, n LA/C&LBR KO = 453, n LA/C, LBR&Emd KO = 300. * p Emd KO = 0.02, remaining p values are non-significant ( p > 0.05; two-sample, two-tailed KS test, Hochberg correction for multiple testing). Numeric values displayed on violin plots represent median values. LA/C lamin A/C, Emd emerin. d Localization of emerin assessed by immunofluorescence microscopy in control and lamin A/C-LBR double knockout myoblasts. Scale bars, 10 μm. Images processed with Fiji. Source data are provided as a file.

Article Snippet: To generate MyoD1 and Pax7 reporter cell lines, C2C12 mouse myoblasts were transfected with the vector pSpCas9(BB)-2A-GFP (pX458, plasmid #48138, Addgene) containing an sgRNA targeting a region 1 kb downstream of MyoD1 or Pax7 , as well as a repair template construct carrying the 64xLacO repeats flanked by regions homologous to MyoD1 and Pax7 , respectively (see also , chapter “Generation of repair template vector” and Supplementary Table for primer sequences).

Techniques: Western Blot, Knock-Out, Clone Assay, Two Tailed Test, Immunofluorescence, Microscopy, Control, Double Knockout

Journal: Nature Communications

Article Title: MyoD1 localization at the nuclear periphery is mediated by association of WFS1 with active enhancers

doi: 10.1038/s41467-025-57758-x

Figure Lengend Snippet: a Immunostaining and 3D reconstruction of H3K9me3-marked heterochromatin in wildtype and lamin A/C-LBR double knockout C2C12 cells. Scale bars, 10 μm. b Graphs show quantification of mean area per intranuclear H3K9me3-positive spot (left), and number of intranuclear foci per cell (right). Bar graphs represent mean ± SD. n Wt = 40, n KO = 31, n KO2 = 31 (single data points are displayed). Left graph: H(2) = 11.72, ** p KO = 0.0096, ** p KO2 = 0.0058; right graph: H(2) = 52.07, **** p KO = 9.8 × 10 -8 , **** p KO2 = 6.6 × 10 −11 (Kruskal-Wallis test). c Nucleoplasmic over peripheral H3K9me3 signal ratio determined using line intensity profiles across the nucleus in wildtype and lamin A/C-LBR double knockout cells. Line graph on the right shows a representative H3K9me3 fluorescence intensity plot measured along the dashed lines in the images shown in ( a ). Bar graph displays mean values ± SD ( n Wt = 4, n KO = 5). t (7) = 8.31, **** p = 7.1 × 10 −5 (two-tailed t-test). d Lamin B1 immunostaining of indicated cell lines. Scale bars, 10 μm. e Lamin B1 ChIP-qPCR analyses demonstrating loss of lamin B1 binding to LADs at the nuclear periphery of lamin A/C-LBR double knockout cells. Data represent mean values ± SEM of three biological replicates. Horizontal dashed line indicates the signal obtained using unspecific IgG antibodies in control ChIP. The genomic positions of tested LADs are indicated in the table. f Representative confocal images of fluorescence in-situ hybridization (FISH) signal using a GFP-labeled BAC probe for a LAD region (green dots). Dashed line indicates nuclear border. Scale bars, 10 μm. g Radial distance of LAD region to the nuclear periphery upon depletion of lamin A/C and LBR as detected by FISH. n Wt = 909, n KO = 666, **** p = 7.6 × 10 -4 (two-sample, two-tailed KS test). Images in ( a ) processed with Imaris, and in ( d ) and ( f ) with Fiji. Source data are provided as a file.

Article Snippet: To generate MyoD1 and Pax7 reporter cell lines, C2C12 mouse myoblasts were transfected with the vector pSpCas9(BB)-2A-GFP (pX458, plasmid #48138, Addgene) containing an sgRNA targeting a region 1 kb downstream of MyoD1 or Pax7 , as well as a repair template construct carrying the 64xLacO repeats flanked by regions homologous to MyoD1 and Pax7 , respectively (see also , chapter “Generation of repair template vector” and Supplementary Table for primer sequences).

Techniques: Immunostaining, Double Knockout, Fluorescence, Two Tailed Test, ChIP-qPCR, Binding Assay, Control, In Situ Hybridization, Labeling

Journal: Nature Communications

Article Title: MyoD1 localization at the nuclear periphery is mediated by association of WFS1 with active enhancers

doi: 10.1038/s41467-025-57758-x

Figure Lengend Snippet: a Schematic overview of the strategy for generating multiple protein knockout cell lines. MyoD1 reporter C2C12 myoblasts constitutively expressing Cas9 were transfected with three synthetic sgRNAs targeting the gene of interest. Depletion of various candidate proteins was performed consecutively. b Detection of normalized radial distance of MyoD1 to periphery in proliferating myoblasts following depletion of indicated protein(s). Violin plots were generated from at least 450 data points. n Wt = 722, n 4KO = 454, n 3KO = 898, n 2KO = 685, n NET39 KO = 500; n Tmem38a KO = 571, n WFS1 KO = 517. *** p 4KO = 2 × 10 −4 , **** p 3KO = 3.2 × 10 −6 , **** p 2KO = 7.9 × 10 −5 , p NET39 KO = 0.6; *** p Tmem38a KO = 2 × 10 −4 , **** p WFS1 KO = 1 × 10 −6 (two-sample two-tailed KS test, Hochberg correction for multiple testing). Numeric values displayed on the violin plot represent median values. Source data are provided as a file.

Article Snippet: To generate MyoD1 and Pax7 reporter cell lines, C2C12 mouse myoblasts were transfected with the vector pSpCas9(BB)-2A-GFP (pX458, plasmid #48138, Addgene) containing an sgRNA targeting a region 1 kb downstream of MyoD1 or Pax7 , as well as a repair template construct carrying the 64xLacO repeats flanked by regions homologous to MyoD1 and Pax7 , respectively (see also , chapter “Generation of repair template vector” and Supplementary Table for primer sequences).

Techniques: Knock-Out, Expressing, Transfection, Generated, Two Tailed Test