myotube differentiation  (ATCC)


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    Structured Review

    ATCC myotube differentiation
    Functional features of hAFSC-derived MNs. (A) Calcium imaging in hAFSC-derived MNs after 22 days of <t>differentiation.</t> Cells were stimulated with 50 mM of KCl. F/F0 represents the ratio of fluorescence intensity for each cell at the indicated time. (B) NMJ detection in the co-culture of hAFSC-derived MNs and myotubes. MNs were labelled with a blue vital staining, whereas the AChRs on myotubes were stained with the α-BTX (green). Red rectangle represents enlarged area (60x). White arrow indicates neuronal terminals ending on the α-BTX (AChRs). Magnification 40x, scale bar 20 µm. The picture is representative of 3 different experiments. (C) Video analysis of the muscle cell contractions in single culture of myotubes, in a contacting co-culture (hAFSC-derived MNs plus <t>myotube)</t> and in a non-contacting co-culture between hAFSC-derived MNs and myotubes. The movements of different points were tracked and quantified by Celleste Image Analysis Software. Graphs are representative of the distance covered by moving points inside three differently ROI.
    Myotube Differentiation, supplied by ATCC, used in various techniques. Bioz Stars score: 80/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Human mesenchymal amniotic fluid stem cells reveal an unexpected neuronal potential differentiating into functional spinal motor neurons"

    Article Title: Human mesenchymal amniotic fluid stem cells reveal an unexpected neuronal potential differentiating into functional spinal motor neurons

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2022.936990

    Functional features of hAFSC-derived MNs. (A) Calcium imaging in hAFSC-derived MNs after 22 days of differentiation. Cells were stimulated with 50 mM of KCl. F/F0 represents the ratio of fluorescence intensity for each cell at the indicated time. (B) NMJ detection in the co-culture of hAFSC-derived MNs and myotubes. MNs were labelled with a blue vital staining, whereas the AChRs on myotubes were stained with the α-BTX (green). Red rectangle represents enlarged area (60x). White arrow indicates neuronal terminals ending on the α-BTX (AChRs). Magnification 40x, scale bar 20 µm. The picture is representative of 3 different experiments. (C) Video analysis of the muscle cell contractions in single culture of myotubes, in a contacting co-culture (hAFSC-derived MNs plus myotube) and in a non-contacting co-culture between hAFSC-derived MNs and myotubes. The movements of different points were tracked and quantified by Celleste Image Analysis Software. Graphs are representative of the distance covered by moving points inside three differently ROI.
    Figure Legend Snippet: Functional features of hAFSC-derived MNs. (A) Calcium imaging in hAFSC-derived MNs after 22 days of differentiation. Cells were stimulated with 50 mM of KCl. F/F0 represents the ratio of fluorescence intensity for each cell at the indicated time. (B) NMJ detection in the co-culture of hAFSC-derived MNs and myotubes. MNs were labelled with a blue vital staining, whereas the AChRs on myotubes were stained with the α-BTX (green). Red rectangle represents enlarged area (60x). White arrow indicates neuronal terminals ending on the α-BTX (AChRs). Magnification 40x, scale bar 20 µm. The picture is representative of 3 different experiments. (C) Video analysis of the muscle cell contractions in single culture of myotubes, in a contacting co-culture (hAFSC-derived MNs plus myotube) and in a non-contacting co-culture between hAFSC-derived MNs and myotubes. The movements of different points were tracked and quantified by Celleste Image Analysis Software. Graphs are representative of the distance covered by moving points inside three differently ROI.

    Techniques Used: Functional Assay, Derivative Assay, Imaging, Fluorescence, Co-Culture Assay, Staining, Software

    2) Product Images from "A Tead1-Apelin axis directs paracrine communication from myogenic to endothelial cells in skeletal muscle"

    Article Title: A Tead1-Apelin axis directs paracrine communication from myogenic to endothelial cells in skeletal muscle

    Journal: iScience

    doi: 10.1016/j.isci.2022.104589

    Apln, Aplnr, and Tead1 expression dynamics in regenerating skeletal muscle (A and B) Single-cell RNA-sequencing analysis of a notexin injury response in TA muscle adult mice. TA muscle samples from 0, 2, 5, and 7 days post-injury (d.p.i.) with n = 2-3 mice per time-point were analyzed from De Micheli et al. (2020) (A) UMAP projection of scRNAseq data demonstrating cell-type annotations of clusters using markers shown in Figure S3 . (B) Dot plots showing expression of Apln , Aplnr , and Tead1 by cell-type cluster and time-point. Dot size shows the frequency of cells expressing non-zero transcript level. Dot color shows average expression level. (C and D) In vitro expression of Apln and Aplnr protein by immunofluorescence (C) and mRNA by qRT-PCR (D) in C166 endothelial cells (ECs) and C2C12 myotubes differentiated for 8days n = 5 for C166 ECs; n = 4 for C2C12 myotubes. Scale bar, 100 μm. (E–I) Regeneration of TA muscles of adult WT mice after IM injection of glycerol analyzed at 0, 3, 7, and 14 days.p.i. by gene expression microarray and immunohistology. (E) Experimental overview. (F and G) Apln and Aplnr mRNA levels from transcriptomic analyses, normalized and presented as fold-change relative to mean of 0 days.p.i. Data are mean ± SEM of n = 5 (0, 14 days.p.i.) and n = 6 (3, 7 days.p.i.) mice. (H) Representative images of CD31 and Laminin immunostaining in regenerating muscle samples at 0, 3, 7, and 14 days.p.i. Scale bar, 50 μm. (I) Quantification of CD31 + endothelial cells per cross-sectional area. Data are mean ± SEM of n = 5 TA muscles. In (F–G) and (I), p values are reported by two-tailed unpaired t-test compared to 0 days.p.i.; with ∗p
    Figure Legend Snippet: Apln, Aplnr, and Tead1 expression dynamics in regenerating skeletal muscle (A and B) Single-cell RNA-sequencing analysis of a notexin injury response in TA muscle adult mice. TA muscle samples from 0, 2, 5, and 7 days post-injury (d.p.i.) with n = 2-3 mice per time-point were analyzed from De Micheli et al. (2020) (A) UMAP projection of scRNAseq data demonstrating cell-type annotations of clusters using markers shown in Figure S3 . (B) Dot plots showing expression of Apln , Aplnr , and Tead1 by cell-type cluster and time-point. Dot size shows the frequency of cells expressing non-zero transcript level. Dot color shows average expression level. (C and D) In vitro expression of Apln and Aplnr protein by immunofluorescence (C) and mRNA by qRT-PCR (D) in C166 endothelial cells (ECs) and C2C12 myotubes differentiated for 8days n = 5 for C166 ECs; n = 4 for C2C12 myotubes. Scale bar, 100 μm. (E–I) Regeneration of TA muscles of adult WT mice after IM injection of glycerol analyzed at 0, 3, 7, and 14 days.p.i. by gene expression microarray and immunohistology. (E) Experimental overview. (F and G) Apln and Aplnr mRNA levels from transcriptomic analyses, normalized and presented as fold-change relative to mean of 0 days.p.i. Data are mean ± SEM of n = 5 (0, 14 days.p.i.) and n = 6 (3, 7 days.p.i.) mice. (H) Representative images of CD31 and Laminin immunostaining in regenerating muscle samples at 0, 3, 7, and 14 days.p.i. Scale bar, 50 μm. (I) Quantification of CD31 + endothelial cells per cross-sectional area. Data are mean ± SEM of n = 5 TA muscles. In (F–G) and (I), p values are reported by two-tailed unpaired t-test compared to 0 days.p.i.; with ∗p

    Techniques Used: Expressing, RNA Sequencing Assay, Mouse Assay, In Vitro, Immunofluorescence, Quantitative RT-PCR, Injection, Microarray, Immunostaining, Two Tailed Test

    Myogenic Tead1 inhibits myogenic-endothelial cross-talk (A-C) Co-culture of GFP expressing C166 ECs with myotubes derived from C2C12 myoblasts transfected with scrambled control or Tead1 targeted siRNAs for 3days (B) Representative images of C166-GFP EC and C2C12 co-cultures at 6days, with DAPI counter-stain. Scale bar, 100 μm. (C) Quantification of the total GFP + cell area relative to the total image area. (n = 24 cell culture replicates per group). (D and E) Culture of C166 ECs in presence of conditioned medium harvested from myotubes derived from C2C12 myoblasts transfected with scrambled control or Tead1 targeted siRNAs. Conditioned media were collected from myotube cultures and applied daily to C166 EC cultures for 3 days. (E) Quantification of the number of C166 ECs per well in conditioned medium from control and Tead1 siRNA treated C2C12 myotubes. (n = 49 wells cell culture replicates per group). (F-I) Co-culture of GFP expressing C166 ECs with myotubes derived from C2C12 myoblasts transfected with scrambled control or Tead1 targeted siRNAs in presence of Aplnr siRNAs or Aplnr inhibitor (ML221). (G) Aplnr expression after the transfection of siRNAs targeted Aplnr compared to si-scrambled control (n = 16 per group). (H) Quantification of the total GFP + cell area relative to total image area after si Aplnr k/d (n = 24 per group). (I) Quantification of the total GFP + cell area relative to total image area after 10μM ML221 (Aplnr inhibitor) compared to DMSO control (n = 24 per group). (J–M) Analysis of MCK-Tead1 overexpressing mice following cardiotoxin-induced injury in TA muscle compared to WT controls. (K) Expression of Pecam1 , Icam1 , and Tek (Tie2) mRNA by RT-qPCR in tibialis anterior muscles isolated from adult myofiber-specific Tead1-overexpressing mice (MCK-OE-Tead1) compared to WT C57BL6 controls. (n = 4 mice per group). (L) Representative images of CD31 immunostaining in CTX-injured TA muscles at 3, 7 days.p.i. Scale bar, 50 μm. (M) Quantification of CD31 + endothelial cells at non-injured, 3 and 7 days.p.i. in regenerating regions (n = three to four TA muscles per group). All graphs are reported as mean ± SEM and p values are reported from two-tailed, unpaired t-test between the conditions.
    Figure Legend Snippet: Myogenic Tead1 inhibits myogenic-endothelial cross-talk (A-C) Co-culture of GFP expressing C166 ECs with myotubes derived from C2C12 myoblasts transfected with scrambled control or Tead1 targeted siRNAs for 3days (B) Representative images of C166-GFP EC and C2C12 co-cultures at 6days, with DAPI counter-stain. Scale bar, 100 μm. (C) Quantification of the total GFP + cell area relative to the total image area. (n = 24 cell culture replicates per group). (D and E) Culture of C166 ECs in presence of conditioned medium harvested from myotubes derived from C2C12 myoblasts transfected with scrambled control or Tead1 targeted siRNAs. Conditioned media were collected from myotube cultures and applied daily to C166 EC cultures for 3 days. (E) Quantification of the number of C166 ECs per well in conditioned medium from control and Tead1 siRNA treated C2C12 myotubes. (n = 49 wells cell culture replicates per group). (F-I) Co-culture of GFP expressing C166 ECs with myotubes derived from C2C12 myoblasts transfected with scrambled control or Tead1 targeted siRNAs in presence of Aplnr siRNAs or Aplnr inhibitor (ML221). (G) Aplnr expression after the transfection of siRNAs targeted Aplnr compared to si-scrambled control (n = 16 per group). (H) Quantification of the total GFP + cell area relative to total image area after si Aplnr k/d (n = 24 per group). (I) Quantification of the total GFP + cell area relative to total image area after 10μM ML221 (Aplnr inhibitor) compared to DMSO control (n = 24 per group). (J–M) Analysis of MCK-Tead1 overexpressing mice following cardiotoxin-induced injury in TA muscle compared to WT controls. (K) Expression of Pecam1 , Icam1 , and Tek (Tie2) mRNA by RT-qPCR in tibialis anterior muscles isolated from adult myofiber-specific Tead1-overexpressing mice (MCK-OE-Tead1) compared to WT C57BL6 controls. (n = 4 mice per group). (L) Representative images of CD31 immunostaining in CTX-injured TA muscles at 3, 7 days.p.i. Scale bar, 50 μm. (M) Quantification of CD31 + endothelial cells at non-injured, 3 and 7 days.p.i. in regenerating regions (n = three to four TA muscles per group). All graphs are reported as mean ± SEM and p values are reported from two-tailed, unpaired t-test between the conditions.

    Techniques Used: Co-Culture Assay, Expressing, Derivative Assay, Transfection, Staining, Cell Culture, Mouse Assay, Quantitative RT-PCR, Isolation, Immunostaining, Two Tailed Test

    Apelin is repressed by Tead1 in muscle cells in vitro and in vivo (A) Immunostaining of Apln protein during C2C12 myotube differentiation. Scale bars, 100 μm. (B) Quantification of Apln mRNA by RT-qPCR during C2C12 myotube differentiation relative to Hprt using 2 –dCt method. n = 4 cell culture replicates per time point. (C and D) Quantification of apelin mRNA by RT-qPCR (C) or apelin peptide in supernatant by ELISA (D) in C2C12 myoblasts transfected with scrambled control or Tead1 targeted siRNAs for 3days n = 16-20 replicates per condition. (E–I) Analysis of Tead1 and in adult mice over-expression Tead1 in skeletal muscle myofibers under the muscle creatine kinase (MCK) promoter (MCK-OE-Tead1 mice), compared to WT C57BL6 controls. (F and G) Tead1 mRNA (F) and Apln mRNA (G) expression levels were measured by RT-qPCR and normalized to Hprt in tibialis anterior (TA) muscles. n = 6 mice per condition. (H and I) Apln peptide concentration measured by ELISA in TA muscles (H) or serum (I). n = 5 mice per condition for TA; n = 11 mice per condition for serum. All data are presented as mean ± SEM, and p values are reported from two-tailed, unpaired t-tests between conditions. ∗p
    Figure Legend Snippet: Apelin is repressed by Tead1 in muscle cells in vitro and in vivo (A) Immunostaining of Apln protein during C2C12 myotube differentiation. Scale bars, 100 μm. (B) Quantification of Apln mRNA by RT-qPCR during C2C12 myotube differentiation relative to Hprt using 2 –dCt method. n = 4 cell culture replicates per time point. (C and D) Quantification of apelin mRNA by RT-qPCR (C) or apelin peptide in supernatant by ELISA (D) in C2C12 myoblasts transfected with scrambled control or Tead1 targeted siRNAs for 3days n = 16-20 replicates per condition. (E–I) Analysis of Tead1 and in adult mice over-expression Tead1 in skeletal muscle myofibers under the muscle creatine kinase (MCK) promoter (MCK-OE-Tead1 mice), compared to WT C57BL6 controls. (F and G) Tead1 mRNA (F) and Apln mRNA (G) expression levels were measured by RT-qPCR and normalized to Hprt in tibialis anterior (TA) muscles. n = 6 mice per condition. (H and I) Apln peptide concentration measured by ELISA in TA muscles (H) or serum (I). n = 5 mice per condition for TA; n = 11 mice per condition for serum. All data are presented as mean ± SEM, and p values are reported from two-tailed, unpaired t-tests between conditions. ∗p

    Techniques Used: In Vitro, In Vivo, Immunostaining, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, Transfection, Mouse Assay, Over Expression, Expressing, Concentration Assay, Two Tailed Test

    3) Product Images from "Protein Hydrolysate from Spirulina platensis Prevents Dexamethasone-Induced Muscle Atrophy via Akt/Foxo3 Signaling in C2C12 Myotubes"

    Article Title: Protein Hydrolysate from Spirulina platensis Prevents Dexamethasone-Induced Muscle Atrophy via Akt/Foxo3 Signaling in C2C12 Myotubes

    Journal: Marine Drugs

    doi: 10.3390/md20060365

    Effect of Spirulina hydrolysate (SPH) on length and diameter of C2C12 myotubes. Data are expressed as the mean ± standard deviation (SD). * p
    Figure Legend Snippet: Effect of Spirulina hydrolysate (SPH) on length and diameter of C2C12 myotubes. Data are expressed as the mean ± standard deviation (SD). * p

    Techniques Used: Standard Deviation

    Effect of SPH on mRNA expression of ( A ) MyoD1, ( B ) Myf5, and ( C ) myogenin in C2C12 myotubes according to the differentiation duration. Data are expressed as the mean ± SD. ** p
    Figure Legend Snippet: Effect of SPH on mRNA expression of ( A ) MyoD1, ( B ) Myf5, and ( C ) myogenin in C2C12 myotubes according to the differentiation duration. Data are expressed as the mean ± SD. ** p

    Techniques Used: Expressing

    Effect of Spirulina hydrolysate (SPH) on ( A ) length and ( B ) diameter of C2C12 myotubes treated with dexamethasone (DEX; 50 nM). Data are expressed as the mean ± SD. *** p
    Figure Legend Snippet: Effect of Spirulina hydrolysate (SPH) on ( A ) length and ( B ) diameter of C2C12 myotubes treated with dexamethasone (DEX; 50 nM). Data are expressed as the mean ± SD. *** p

    Techniques Used:

    Effect of SPH on the protein expression of ( A ) MyoD1 and ( B ) myogenin in C2C12 myotubes. Cells were differentiated for 6 days with SPH treatment. Data are expressed as the mean ± SD. * p
    Figure Legend Snippet: Effect of SPH on the protein expression of ( A ) MyoD1 and ( B ) myogenin in C2C12 myotubes. Cells were differentiated for 6 days with SPH treatment. Data are expressed as the mean ± SD. * p

    Techniques Used: Expressing

    Effect of SPH on the protein expression of ( A ) cytosolic Akt and nuclear ( B ) FoxO3a, ( C ) MuRF-1, and ( D ) Atrogin-1 in C2C12 myotubes treated with DEX (50 nM). Data are expressed as the mean ± SD. * p
    Figure Legend Snippet: Effect of SPH on the protein expression of ( A ) cytosolic Akt and nuclear ( B ) FoxO3a, ( C ) MuRF-1, and ( D ) Atrogin-1 in C2C12 myotubes treated with DEX (50 nM). Data are expressed as the mean ± SD. * p

    Techniques Used: Expressing

    Effect of SPH on mRNA expression of ( A ) Atrogin-1, ( B ) MuRF-1, and ( C ) FoxO3a in C2C12 myotubes treated with dexamethasone (DEX; 50 nM). Data are expressed as the mean ± SD. * p
    Figure Legend Snippet: Effect of SPH on mRNA expression of ( A ) Atrogin-1, ( B ) MuRF-1, and ( C ) FoxO3a in C2C12 myotubes treated with dexamethasone (DEX; 50 nM). Data are expressed as the mean ± SD. * p

    Techniques Used: Expressing

    4) Product Images from "Downregulation of let-7 by Electrical Acupuncture Increases Protein Synthesis in Mice"

    Article Title: Downregulation of let-7 by Electrical Acupuncture Increases Protein Synthesis in Mice

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2021.697139

    Provision of let-7 inhibited Igf-1, Igfrα and Igfrβ in cultured C2C12 myotubes. (A) Total RNA was isolated from C2C12 myotubes transfected with control/mimic, let-7-5p/mimic and let-7/inhibitor. The expression of let-7-5p was assayed by real time qPCR. The bar graph shows let-7-5p from different group compared with levels in control/mimic (defined as onefold). Results are normalized to miR103 (Bars: mean ± SE; n = 4/group; * p
    Figure Legend Snippet: Provision of let-7 inhibited Igf-1, Igfrα and Igfrβ in cultured C2C12 myotubes. (A) Total RNA was isolated from C2C12 myotubes transfected with control/mimic, let-7-5p/mimic and let-7/inhibitor. The expression of let-7-5p was assayed by real time qPCR. The bar graph shows let-7-5p from different group compared with levels in control/mimic (defined as onefold). Results are normalized to miR103 (Bars: mean ± SE; n = 4/group; * p

    Techniques Used: Cell Culture, Isolation, Transfection, Expressing, Real-time Polymerase Chain Reaction

    5) Product Images from "Pyropia yezoensis protein protects against TNF-α-induced myotube atrophy in C2C12 myotubes via the NF-κB signaling pathway"

    Article Title: Pyropia yezoensis protein protects against TNF-α-induced myotube atrophy in C2C12 myotubes via the NF-κB signaling pathway

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2021.12125

    Effects of PYCP on the expression of TNF-R1 in TNF-α-treated C2C12 myotubes. TNF-R1 protein expression levels were detected by western blot analysis. GAPDH was used as an internal standard. Data are presented as the mean ± standard deviation of three independent experiments. *P
    Figure Legend Snippet: Effects of PYCP on the expression of TNF-R1 in TNF-α-treated C2C12 myotubes. TNF-R1 protein expression levels were detected by western blot analysis. GAPDH was used as an internal standard. Data are presented as the mean ± standard deviation of three independent experiments. *P

    Techniques Used: Expressing, Western Blot, Standard Deviation

    Effects of PYCP on the production of IL-6 in TNF-α-treated C2C12 myotubes. C2C12 myotubes were treated with 20 ng/ml TNF-α and PYCP (25, 50, or 100 µg/ml) for 48 h. IL-6 in the culture media was measured using ELISA. Data are presented as the mean ± standard deviation of three independent experiments. *P
    Figure Legend Snippet: Effects of PYCP on the production of IL-6 in TNF-α-treated C2C12 myotubes. C2C12 myotubes were treated with 20 ng/ml TNF-α and PYCP (25, 50, or 100 µg/ml) for 48 h. IL-6 in the culture media was measured using ELISA. Data are presented as the mean ± standard deviation of three independent experiments. *P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Standard Deviation

    Effects of PYCP on the ubiquitin-proteasome system in TNF-α-treated C2C12 myotubes. (A) The atrogin-1/MAFbx and MuRF1 protein expression levels were detected by western blot analysis. GAPDH was used as an internal standard. (B) 20S proteasome activity was assessed by detecting AMC in cell lysates after cleavage from the AMC-tagged peptide LLVY. Data are presented as the mean ± standard deviation of three independent experiments. *P
    Figure Legend Snippet: Effects of PYCP on the ubiquitin-proteasome system in TNF-α-treated C2C12 myotubes. (A) The atrogin-1/MAFbx and MuRF1 protein expression levels were detected by western blot analysis. GAPDH was used as an internal standard. (B) 20S proteasome activity was assessed by detecting AMC in cell lysates after cleavage from the AMC-tagged peptide LLVY. Data are presented as the mean ± standard deviation of three independent experiments. *P

    Techniques Used: Expressing, Western Blot, Activity Assay, Standard Deviation

    Effects of TNF-α and PYCP on the cytotoxicity of C2C12 myotubes. (A) The cell viability of C2C12 myotubes in the presence of 0, 12.5, 25, 50, and 100 µg/ml PYCP for 24 h. (B) The cell viability of C2C12 myotubes treated with 25, 50, and 100 µg/ml PYCP for 24 h and cotreated with 20 ng/ml TNF-α for 24 h.*P
    Figure Legend Snippet: Effects of TNF-α and PYCP on the cytotoxicity of C2C12 myotubes. (A) The cell viability of C2C12 myotubes in the presence of 0, 12.5, 25, 50, and 100 µg/ml PYCP for 24 h. (B) The cell viability of C2C12 myotubes treated with 25, 50, and 100 µg/ml PYCP for 24 h and cotreated with 20 ng/ml TNF-α for 24 h.*P

    Techniques Used:

    Effects of PYCP on the expression of MyoD and myogenin in TNF-α-treated C2C12 myotubes. MyoD and myogenin protein expression levels were detected by western blot analysis. GAPDH was used as an internal standard. Data are presented as the mean ± standard deviation of three independent experiments. *P
    Figure Legend Snippet: Effects of PYCP on the expression of MyoD and myogenin in TNF-α-treated C2C12 myotubes. MyoD and myogenin protein expression levels were detected by western blot analysis. GAPDH was used as an internal standard. Data are presented as the mean ± standard deviation of three independent experiments. *P

    Techniques Used: Expressing, Western Blot, Standard Deviation

    Effects of PYCP on the production of intracellular ROS in TNF-α-treated C2C12 myotubes. C2C12 myotubes were treated with 20 ng/ml TNF-α and PYCP (25, 50, or 100 µg/ml) for 48 h. Intracellular ROS production was measured using DCF-DA and fluorescence intensity analysis. Data are presented as the mean ± standard deviation of three independent experiments. *P
    Figure Legend Snippet: Effects of PYCP on the production of intracellular ROS in TNF-α-treated C2C12 myotubes. C2C12 myotubes were treated with 20 ng/ml TNF-α and PYCP (25, 50, or 100 µg/ml) for 48 h. Intracellular ROS production was measured using DCF-DA and fluorescence intensity analysis. Data are presented as the mean ± standard deviation of three independent experiments. *P

    Techniques Used: Fluorescence, Standard Deviation

    Effects of PYCP on the activation and translocation of NF-κB/p65 in TNF-α-treated C2C12 myotubes. (A) p-IκBα, IκBα, and NF-κB/p65 protein expression levels of cytosolic fractions were detected by western blot analysis. (B) NF-κB/p65 levels were measured in the nuclear fractions by western blot analysis. β-actin and lamin B1 were used as internal controls for the cytosolic and nuclear fractions, respectively. Data are presented as the mean ± standard deviation of three independent experiments. *P
    Figure Legend Snippet: Effects of PYCP on the activation and translocation of NF-κB/p65 in TNF-α-treated C2C12 myotubes. (A) p-IκBα, IκBα, and NF-κB/p65 protein expression levels of cytosolic fractions were detected by western blot analysis. (B) NF-κB/p65 levels were measured in the nuclear fractions by western blot analysis. β-actin and lamin B1 were used as internal controls for the cytosolic and nuclear fractions, respectively. Data are presented as the mean ± standard deviation of three independent experiments. *P

    Techniques Used: Activation Assay, Translocation Assay, Expressing, Western Blot, Standard Deviation

    Effects of PYCP on myotube diameter in TNF-α-treated C2C12 myotubes. Representative images and quantification of myotube diameters are shown for C2C12 myotubes treated with 20 ng/ml TNF-α and PYCP (25, 50, or 100 µg/ml) for 48 h. Images were captured at ×20 magnification (scale bar, 50 µm). Data are presented as the mean ± standard deviation of three independent experiments. *P
    Figure Legend Snippet: Effects of PYCP on myotube diameter in TNF-α-treated C2C12 myotubes. Representative images and quantification of myotube diameters are shown for C2C12 myotubes treated with 20 ng/ml TNF-α and PYCP (25, 50, or 100 µg/ml) for 48 h. Images were captured at ×20 magnification (scale bar, 50 µm). Data are presented as the mean ± standard deviation of three independent experiments. *P

    Techniques Used: Standard Deviation

    6) Product Images from "Microtubule-based Transport is Essential to Distribute RNA and Nascent Protein in Skeletal Muscle"

    Article Title: Microtubule-based Transport is Essential to Distribute RNA and Nascent Protein in Skeletal Muscle

    Journal: bioRxiv

    doi: 10.1101/2021.02.26.433059

    RNAs Exhibit Restricted Diffusion and Directed Transport in Myotubes A) Schematic describing MS2 live cell RNA imaging strategy. B) Example image of a myotube expressing MS2-labeled RNA (top) and RNA trajectories (bottom) from a 53-second imaging time course. Scale bars: 2 µm. See also Video S4. C) Trajectories were categorized according to their motion (see Methods). See also Video S5. D) Diffusion coefficients calculated for “low mobility” (red) and “high mobility” (green) diffusive tracks (left, see Methods), and example tracks from both groups (right). X-axis ticks for scale: 0.5 µm. E) Distance (left) and velocity (right) calculated for “processive” runs (blue) and “crawling” trajectories (purple, see Methods), and example tracks from both groups (far right). X-axis ticks for scale: 0.5 µm. F) Series of images showing RNA particle splitting and merging events from movies analyzed in A-E. Colored triangles denote particle identities. Scale bars: 1 µm. See also Video S6. G) Example RNA tracks from a myotube expressing MS2-labeled RNA during a 50-minute imaging time course. Scale bars: 2 µm. See also Video S7. H) Maximum distance traveled for each track from the 50-minute (red) and 2-minute (blue) imaging time courses. Tracks below dotted line (1 µm) are categorized as stationary. I) Fraction of stationary tracks in 50-minute and 2-minute time courses.
    Figure Legend Snippet: RNAs Exhibit Restricted Diffusion and Directed Transport in Myotubes A) Schematic describing MS2 live cell RNA imaging strategy. B) Example image of a myotube expressing MS2-labeled RNA (top) and RNA trajectories (bottom) from a 53-second imaging time course. Scale bars: 2 µm. See also Video S4. C) Trajectories were categorized according to their motion (see Methods). See also Video S5. D) Diffusion coefficients calculated for “low mobility” (red) and “high mobility” (green) diffusive tracks (left, see Methods), and example tracks from both groups (right). X-axis ticks for scale: 0.5 µm. E) Distance (left) and velocity (right) calculated for “processive” runs (blue) and “crawling” trajectories (purple, see Methods), and example tracks from both groups (far right). X-axis ticks for scale: 0.5 µm. F) Series of images showing RNA particle splitting and merging events from movies analyzed in A-E. Colored triangles denote particle identities. Scale bars: 1 µm. See also Video S6. G) Example RNA tracks from a myotube expressing MS2-labeled RNA during a 50-minute imaging time course. Scale bars: 2 µm. See also Video S7. H) Maximum distance traveled for each track from the 50-minute (red) and 2-minute (blue) imaging time courses. Tracks below dotted line (1 µm) are categorized as stationary. I) Fraction of stationary tracks in 50-minute and 2-minute time courses.

    Techniques Used: Diffusion-based Assay, Imaging, Expressing, Labeling

    Computational Simulation Confirms That Directed Transport Is Required to Disperse mRNA in Myofibers A) Diagram of RNA mobility states modeled in the simulation of RNA motion in myofibers. RNA molecules were spawned from the nuclear envelope and allowed to transition between “low mobility” diffusion and active transport states. While in each state, RNAs moved in 3D space according to parameters sampled from distributions shown in Fig. 6D and 6E obtained from C2C12 myotube imaging. B) Network diagram of the discrete-time Markov chain (DTMC) model developed to simulate RNA generation, motion, and decay. Production and decay rates were constrained by measured mRNA density and half-life (See Fig. 1 and Fig. S5 ). Transition probabilities were derived from the average number of RNAs observed in each state in C2C12 myotubes (See Fig. 6C ). Simulations were conducted in 3D using myofiber and nuclear geometries segmented from confocal images. C) Comparison of RNA localization patterns observed in 1000 hr simulations of Polr2a RNA either with (right) or without (left) directed transport states. The average RNA density was calculated by sampling once every hour, with the first 10% of the simulation discarded as burn-in. See also Video S8. D) Distance to nucleus measured for simulated Polr2a mRNAs (see C) either with (orange) or without (blue) directed transport states. Shown for comparison is the distribution from a simulation of Polr2a mRNAs in the “high mobility” diffusion state (green, see Fig. 6D ) and a null distribution generated from randomly selected cytoplasmic coordinates (grey).
    Figure Legend Snippet: Computational Simulation Confirms That Directed Transport Is Required to Disperse mRNA in Myofibers A) Diagram of RNA mobility states modeled in the simulation of RNA motion in myofibers. RNA molecules were spawned from the nuclear envelope and allowed to transition between “low mobility” diffusion and active transport states. While in each state, RNAs moved in 3D space according to parameters sampled from distributions shown in Fig. 6D and 6E obtained from C2C12 myotube imaging. B) Network diagram of the discrete-time Markov chain (DTMC) model developed to simulate RNA generation, motion, and decay. Production and decay rates were constrained by measured mRNA density and half-life (See Fig. 1 and Fig. S5 ). Transition probabilities were derived from the average number of RNAs observed in each state in C2C12 myotubes (See Fig. 6C ). Simulations were conducted in 3D using myofiber and nuclear geometries segmented from confocal images. C) Comparison of RNA localization patterns observed in 1000 hr simulations of Polr2a RNA either with (right) or without (left) directed transport states. The average RNA density was calculated by sampling once every hour, with the first 10% of the simulation discarded as burn-in. See also Video S8. D) Distance to nucleus measured for simulated Polr2a mRNAs (see C) either with (orange) or without (blue) directed transport states. Shown for comparison is the distribution from a simulation of Polr2a mRNAs in the “high mobility” diffusion state (green, see Fig. 6D ) and a null distribution generated from randomly selected cytoplasmic coordinates (grey).

    Techniques Used: Diffusion-based Assay, Imaging, Derivative Assay, Sampling, Generated

    7) Product Images from "PEG-BHD1028 Peptide Regulates Insulin Resistance and Fatty Acid β-Oxidation, and Mitochondrial Biogenesis by Binding to Two Heterogeneous Binding Sites of Adiponectin Receptors, AdipoR1 and AdipoR2"

    Article Title: PEG-BHD1028 Peptide Regulates Insulin Resistance and Fatty Acid β-Oxidation, and Mitochondrial Biogenesis by Binding to Two Heterogeneous Binding Sites of Adiponectin Receptors, AdipoR1 and AdipoR2

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms22020884

    The regulatory effects of PEG-BHD1028 on mitochondrial biogenesis in C2C12 myotubes. ( A ) The activation of p38 MAPK, PGC-1α, and MKP-1 following the treatment with PEG-BHD1028 was observed by western blot. ( B ) The change in the ratio of the phosphorylation band density of p38 MAPK to control MAPK was quantified. ( C , D ) The quantification of PGC-1α and MKP-1 proteins was represented as a ratio to GAPDH. Results are presented as means ± SEM ( n = 3). ** p ≤ 0.01, * p ≤ 0.05 vs. CTL (Negative control).
    Figure Legend Snippet: The regulatory effects of PEG-BHD1028 on mitochondrial biogenesis in C2C12 myotubes. ( A ) The activation of p38 MAPK, PGC-1α, and MKP-1 following the treatment with PEG-BHD1028 was observed by western blot. ( B ) The change in the ratio of the phosphorylation band density of p38 MAPK to control MAPK was quantified. ( C , D ) The quantification of PGC-1α and MKP-1 proteins was represented as a ratio to GAPDH. Results are presented as means ± SEM ( n = 3). ** p ≤ 0.01, * p ≤ 0.05 vs. CTL (Negative control).

    Techniques Used: Activation Assay, Pyrolysis Gas Chromatography, Western Blot, Negative Control

    The relative protein levels of PPARα activity in differentiated C2C12 myotubes. ( A ) Representative blots illustrating the effect of PEG-BHD1028 on PPARα activation. ( B ) Western blot signals were quantified by a densitometer. The amount of protein was normalized to GAPDH. Results are presented as means ± SEM ( n = 3). ** p ≤ 0.01, * p ≤ 0.05 vs. CTL (Negative control).
    Figure Legend Snippet: The relative protein levels of PPARα activity in differentiated C2C12 myotubes. ( A ) Representative blots illustrating the effect of PEG-BHD1028 on PPARα activation. ( B ) Western blot signals were quantified by a densitometer. The amount of protein was normalized to GAPDH. Results are presented as means ± SEM ( n = 3). ** p ≤ 0.01, * p ≤ 0.05 vs. CTL (Negative control).

    Techniques Used: Activity Assay, Activation Assay, Western Blot, Negative Control

    Immunoblot analysis of AMPK and ACC phosphorylation in C2C12 muscle cells. Differentiated C2C12 myotubes were treated with 20, 100, 500 nM of PEG-BHD1028 and globular adiponectin (5 μg/mL) was treated as a positive control for 30 min. The phosphorylation of AMPK and ACC was analyzed by western blot ( A ). Western blot signals were quantified by a densitometer ( B , C ). Results are presented as means ± standards error of the mean ( n = 3). ** p ≤ 0.01, * p ≤ 0.05 vs. CTL (Negative control).
    Figure Legend Snippet: Immunoblot analysis of AMPK and ACC phosphorylation in C2C12 muscle cells. Differentiated C2C12 myotubes were treated with 20, 100, 500 nM of PEG-BHD1028 and globular adiponectin (5 μg/mL) was treated as a positive control for 30 min. The phosphorylation of AMPK and ACC was analyzed by western blot ( A ). Western blot signals were quantified by a densitometer ( B , C ). Results are presented as means ± standards error of the mean ( n = 3). ** p ≤ 0.01, * p ≤ 0.05 vs. CTL (Negative control).

    Techniques Used: Positive Control, Western Blot, Negative Control

    Glucose uptake enhancement effect of PEG-BHD1028. Effect of insulin on the uptake of 2-deoxy-D-glucose (2DG) in non-resistance ( A ). PEG-BHD1028 ameliorated palmitate-induced insulin resistance in C2C12 myotubes ( B ). Globular adiponectin (5 μg/mL) was treated as a positive control. Results are presented as means ± standards error of the mean ( n = 5). ** p ≤ 0.01, * p ≤ 0.05 vs. CTL (Negative control).
    Figure Legend Snippet: Glucose uptake enhancement effect of PEG-BHD1028. Effect of insulin on the uptake of 2-deoxy-D-glucose (2DG) in non-resistance ( A ). PEG-BHD1028 ameliorated palmitate-induced insulin resistance in C2C12 myotubes ( B ). Globular adiponectin (5 μg/mL) was treated as a positive control. Results are presented as means ± standards error of the mean ( n = 5). ** p ≤ 0.01, * p ≤ 0.05 vs. CTL (Negative control).

    Techniques Used: Positive Control, Negative Control

    Effect of PEG-BHD1028 on cytotoxicity in C2C12 myotubes. C2C12 myotubes were treated with the serial concentrations of PEG-BHD1028 ranging from 7.8 nM to 1 mM for 24 h. The results are shown as a mean ± standards error of the mean ( n = 3) relative to the control samples. ** p ≤ 0.01 vs. CLT (Negative control).
    Figure Legend Snippet: Effect of PEG-BHD1028 on cytotoxicity in C2C12 myotubes. C2C12 myotubes were treated with the serial concentrations of PEG-BHD1028 ranging from 7.8 nM to 1 mM for 24 h. The results are shown as a mean ± standards error of the mean ( n = 3) relative to the control samples. ** p ≤ 0.01 vs. CLT (Negative control).

    Techniques Used: Negative Control

    PEG-BHD1028 increases mitochondria-related gene expression in C2C12 myotubes. mRNA expressions of COX2, ND5 and PGC-1α were quantified using qPCR in C2C12 myotubes. The threshold cycle ( C t ) for each reaction was normalized (Δ C t ) by the value of the β-Actin. The value of Δ C t was further normalized to exhibit the comparative expression levels with respect to the mean value. Results are presented as means ± SEM ( n = 3). *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 vs. Negative control.
    Figure Legend Snippet: PEG-BHD1028 increases mitochondria-related gene expression in C2C12 myotubes. mRNA expressions of COX2, ND5 and PGC-1α were quantified using qPCR in C2C12 myotubes. The threshold cycle ( C t ) for each reaction was normalized (Δ C t ) by the value of the β-Actin. The value of Δ C t was further normalized to exhibit the comparative expression levels with respect to the mean value. Results are presented as means ± SEM ( n = 3). *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 vs. Negative control.

    Techniques Used: Expressing, Pyrolysis Gas Chromatography, Real-time Polymerase Chain Reaction, Negative Control

    PEG-BHD1028 increases mitochondrial biogenesis in C2C12 myotubes. C2C12 myotubes were treated with 20, 100, and 500 nM of PEG-BHD1028 and 5 μg/mL of gAD for 24 h. Mitotracker-stained mitochondrial intensity of cells ( A ) was measured using a confocal fluorescence microscope, and the relative mitotracker intensity against nuclear DAPI intensity ( B ) was calculated. Each fluorescence intensity excluding background of 5 spots of 82 mm 2 in the same area was obtained. The intensity of each cell was normalized reflecting the confluence coefficient. Results are presents as means ± SEM ( n = 5). *** p ≤ 0.0001 vs. CTL (Negative control).
    Figure Legend Snippet: PEG-BHD1028 increases mitochondrial biogenesis in C2C12 myotubes. C2C12 myotubes were treated with 20, 100, and 500 nM of PEG-BHD1028 and 5 μg/mL of gAD for 24 h. Mitotracker-stained mitochondrial intensity of cells ( A ) was measured using a confocal fluorescence microscope, and the relative mitotracker intensity against nuclear DAPI intensity ( B ) was calculated. Each fluorescence intensity excluding background of 5 spots of 82 mm 2 in the same area was obtained. The intensity of each cell was normalized reflecting the confluence coefficient. Results are presents as means ± SEM ( n = 5). *** p ≤ 0.0001 vs. CTL (Negative control).

    Techniques Used: Staining, Fluorescence, Microscopy, Negative Control

    8) Product Images from "BAM15‐mediated mitochondrial uncoupling protects against obesity and improves glycemic control"

    Article Title: BAM15‐mediated mitochondrial uncoupling protects against obesity and improves glycemic control

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.202012088

    BAM15 stimulates insulin signaling and oxidation of glucose and fatty acids in an AMPK‐dependent manner (A) Representative immunoblots and (B) quantitative analysis of AKT (1 μM insulin: P = 0.038), (C) AS160 (‐ insulin: P = 0.036, 1 μM insulin: P = 0.0038), and (D) AMPK (‐ insulin: P = 0.018) phosphorylation ± 16‐h BAM15 treatment and 15‐min insulin stimulation ( N = 3 per condition) in C2C12 myotubes. (E) Representative immunoblots and quantitative analysis of (F) plasma membrane GLUT4 translocation ( P = 0.0139) ± 16‐h BAM15 treatment ( N = 3 per condition) in C2C12 myotubes. (G) [3‐ 3 H]glucose uptake ± 16‐h BAM15 treatment and 30‐min insulin stimulation ( N = 4 per condition; vehicle versus vehicle + insulin: P = 0.0016, vehicle versus BAM15: P
    Figure Legend Snippet: BAM15 stimulates insulin signaling and oxidation of glucose and fatty acids in an AMPK‐dependent manner (A) Representative immunoblots and (B) quantitative analysis of AKT (1 μM insulin: P = 0.038), (C) AS160 (‐ insulin: P = 0.036, 1 μM insulin: P = 0.0038), and (D) AMPK (‐ insulin: P = 0.018) phosphorylation ± 16‐h BAM15 treatment and 15‐min insulin stimulation ( N = 3 per condition) in C2C12 myotubes. (E) Representative immunoblots and quantitative analysis of (F) plasma membrane GLUT4 translocation ( P = 0.0139) ± 16‐h BAM15 treatment ( N = 3 per condition) in C2C12 myotubes. (G) [3‐ 3 H]glucose uptake ± 16‐h BAM15 treatment and 30‐min insulin stimulation ( N = 4 per condition; vehicle versus vehicle + insulin: P = 0.0016, vehicle versus BAM15: P

    Techniques Used: Western Blot, Translocation Assay

    9) Product Images from "BAM15‐mediated mitochondrial uncoupling protects against obesity and improves glycemic control"

    Article Title: BAM15‐mediated mitochondrial uncoupling protects against obesity and improves glycemic control

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.202012088

    BAM15 stimulates insulin signaling and oxidation of glucose and fatty acids in an AMPK‐dependent manner (A) Representative immunoblots and (B) quantitative analysis of AKT (1 μM insulin: P = 0.038), (C) AS160 (‐ insulin: P = 0.036, 1 μM insulin: P = 0.0038), and (D) AMPK (‐ insulin: P = 0.018) phosphorylation ± 16‐h BAM15 treatment and 15‐min insulin stimulation ( N = 3 per condition) in C2C12 myotubes. (E) Representative immunoblots and quantitative analysis of (F) plasma membrane GLUT4 translocation ( P = 0.0139) ± 16‐h BAM15 treatment ( N = 3 per condition) in C2C12 myotubes. (G) [3‐ 3 H]glucose uptake ± 16‐h BAM15 treatment and 30‐min insulin stimulation ( N = 4 per condition; vehicle versus vehicle + insulin: P = 0.0016, vehicle versus BAM15: P
    Figure Legend Snippet: BAM15 stimulates insulin signaling and oxidation of glucose and fatty acids in an AMPK‐dependent manner (A) Representative immunoblots and (B) quantitative analysis of AKT (1 μM insulin: P = 0.038), (C) AS160 (‐ insulin: P = 0.036, 1 μM insulin: P = 0.0038), and (D) AMPK (‐ insulin: P = 0.018) phosphorylation ± 16‐h BAM15 treatment and 15‐min insulin stimulation ( N = 3 per condition) in C2C12 myotubes. (E) Representative immunoblots and quantitative analysis of (F) plasma membrane GLUT4 translocation ( P = 0.0139) ± 16‐h BAM15 treatment ( N = 3 per condition) in C2C12 myotubes. (G) [3‐ 3 H]glucose uptake ± 16‐h BAM15 treatment and 30‐min insulin stimulation ( N = 4 per condition; vehicle versus vehicle + insulin: P = 0.0016, vehicle versus BAM15: P

    Techniques Used: Western Blot, Translocation Assay

    10) Product Images from "Cell-Derived Vesicles as TRPC1 Channel Delivery Systems for the Recovery of Cellular Respiratory and Proliferative Capacities"

    Article Title: Cell-Derived Vesicles as TRPC1 Channel Delivery Systems for the Recovery of Cellular Respiratory and Proliferative Capacities

    Journal: bioRxiv

    doi: 10.1101/2020.05.16.099283

    (A) CDV yield is influenced by cell confluence and cellular differentiation status. Vesicle yield increased with increasing confluence and in differentiated myotubes (left). Mean CDV diameter was not statistically different in samples generated from myoblasts or myotubes (right) (n.s.: not significant). (B) CDV size distribution without (left) and with extrusion (right) through 200 nm diameter pores. (C) Freeze fracture SEM of a non-extruded CDV sample. Asterisks (*) identify liposomes that partially look out of the surface, hashtags (#) depict imprints within the surface from CDVs that were entirely detached during the fracture process thereby leaving a crater in the surface. Arrows reveal smaller liposomes that were enclosed within the lumens of larger liposomes during the CDV formation. Scale bar: 1 µm. (D) TEM micrograph of extruded CDVs shown in B (right). The blue colored area depicts the free carbon grid. The black triangles mark the lipid bilayer with a diameter of 4 nm to 5 nm; individual monolayers are visible as thin dark lines. The 2 CDVs adjacent to the free carbon grid are deformed due to the limited height of the sample holder. Scale bar: 100 nm.
    Figure Legend Snippet: (A) CDV yield is influenced by cell confluence and cellular differentiation status. Vesicle yield increased with increasing confluence and in differentiated myotubes (left). Mean CDV diameter was not statistically different in samples generated from myoblasts or myotubes (right) (n.s.: not significant). (B) CDV size distribution without (left) and with extrusion (right) through 200 nm diameter pores. (C) Freeze fracture SEM of a non-extruded CDV sample. Asterisks (*) identify liposomes that partially look out of the surface, hashtags (#) depict imprints within the surface from CDVs that were entirely detached during the fracture process thereby leaving a crater in the surface. Arrows reveal smaller liposomes that were enclosed within the lumens of larger liposomes during the CDV formation. Scale bar: 1 µm. (D) TEM micrograph of extruded CDVs shown in B (right). The blue colored area depicts the free carbon grid. The black triangles mark the lipid bilayer with a diameter of 4 nm to 5 nm; individual monolayers are visible as thin dark lines. The 2 CDVs adjacent to the free carbon grid are deformed due to the limited height of the sample holder. Scale bar: 100 nm.

    Techniques Used: Cell Differentiation, Generated, Transmission Electron Microscopy

    (A) Proteome analysis of CDVs and its comparison to published data. [ 31 ] Differences in GO-assigned fractions of identified proteins in between CDVs and C2C12 derived from myotubes. CDVs exhibit relative enrichments of protein identifications with GO terms associated with membrane and the extracellular region. (B) The percentage of total identified proteins for GOs of CDV samples provides an overview of the cell component protein associations. (C) Chromatographic traces (PRM measurements) of fragment ions matching proteotypic peptides from the respective calcium channels TRPC1, ORAI1, and TRPA1. Grey asterisks mark the relevant signals. (D) Lipid abundances for CDVs and C2C12 myotubes. (E) Comparison of lipid abundances between CDVs and C2C12 myotubes. The most significant changes in lipid content (CDVs relative myotubes) originates from the sphingomyelins (+5.99%) and phosphatidylserines (+12.5%) as well as from phosphatidylcholines (−18.51%). A list of the detailed concentrations of lipid components in CDVs and whole cell samples is available in the appendix. Lipid abbreviations are: TG: triglyceride, SM: sphingomyelin, PS: phosphatidylserine, PI: phosphatidylinositol, PE: phosphatidylethanolamine, PC: phosphatidylcholine, LPC: lysophosphatidylcholine, CER: ceramide.
    Figure Legend Snippet: (A) Proteome analysis of CDVs and its comparison to published data. [ 31 ] Differences in GO-assigned fractions of identified proteins in between CDVs and C2C12 derived from myotubes. CDVs exhibit relative enrichments of protein identifications with GO terms associated with membrane and the extracellular region. (B) The percentage of total identified proteins for GOs of CDV samples provides an overview of the cell component protein associations. (C) Chromatographic traces (PRM measurements) of fragment ions matching proteotypic peptides from the respective calcium channels TRPC1, ORAI1, and TRPA1. Grey asterisks mark the relevant signals. (D) Lipid abundances for CDVs and C2C12 myotubes. (E) Comparison of lipid abundances between CDVs and C2C12 myotubes. The most significant changes in lipid content (CDVs relative myotubes) originates from the sphingomyelins (+5.99%) and phosphatidylserines (+12.5%) as well as from phosphatidylcholines (−18.51%). A list of the detailed concentrations of lipid components in CDVs and whole cell samples is available in the appendix. Lipid abbreviations are: TG: triglyceride, SM: sphingomyelin, PS: phosphatidylserine, PI: phosphatidylinositol, PE: phosphatidylethanolamine, PC: phosphatidylcholine, LPC: lysophosphatidylcholine, CER: ceramide.

    Techniques Used: Derivative Assay

    11) Product Images from "A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90"

    Article Title: A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90

    Journal: Autophagy

    doi: 10.4161/15548627.2014.994402

    eATP induces P2RX7-mediated LP formation and autophagy in dystrophic myotubes. ( A ) EtBr fluorescence in Wt, Dmd mdx , and Dmd mdx p2rx7 −/− myotubes following 30 min exposure to 3 mM eATP with and without preincubation with the autophagy inhibitor 3-MA (5 mM) or with the HSP90 inhibitor geldanamycin (GA, 75 nM). Note the absence of response in Dmd mdx p2rx7 −/− myotubes lacking P2RX7. Digitonin (50 μg/ml) represents permeabilized positive controls. ( B ) Summary data representing EtBr uptake in Wt and Dmd mdx myotubes shown in ( A ). eATP-dependent increases in EtBr fluorescence were observed in both Wt and Dmd mdx myotubes with Dmd mdx displaying significantly higher uptake relative to Wt cells. In both Wt and Dmd mdx myotubes, preincubation with P2RX7 antagonist A438079 (100 μM), 3-MA (5 mM) or GA (75 nM) produced significant reductions in eATP-induced EtBr fluorescence. Three-MA alone blocked EtBr background signal in unstimulated cells. ( C ) Summary data of differences in LDH release from Wt, Dmd mdx and Dmd mdx p2rx7 −/− myoblasts following 30 min exposure to 3 mM eATP, and Wt and Dmd mdx in the absence or presence of autophagy and P2RX7 inhibitors (3-MA [5 mM] and A804598 [100 nM], respectively). ( D ) Representative western blots showing P2RX7 and LC3-II levels in Wt, Dmd mdx and Dmd mdx p2rx7 −/− double-mutant myotubes incubated for 30 min with 1 mM BzATP. Graph depicts fold change in LC3-II levels in Dmd mdx and Dmd mdx p2rx7 −/− myotubes compared to Wt myotubes following 30 min exposure to 1 mM BzATP. Means +/− SE, n = 5, P
    Figure Legend Snippet: eATP induces P2RX7-mediated LP formation and autophagy in dystrophic myotubes. ( A ) EtBr fluorescence in Wt, Dmd mdx , and Dmd mdx p2rx7 −/− myotubes following 30 min exposure to 3 mM eATP with and without preincubation with the autophagy inhibitor 3-MA (5 mM) or with the HSP90 inhibitor geldanamycin (GA, 75 nM). Note the absence of response in Dmd mdx p2rx7 −/− myotubes lacking P2RX7. Digitonin (50 μg/ml) represents permeabilized positive controls. ( B ) Summary data representing EtBr uptake in Wt and Dmd mdx myotubes shown in ( A ). eATP-dependent increases in EtBr fluorescence were observed in both Wt and Dmd mdx myotubes with Dmd mdx displaying significantly higher uptake relative to Wt cells. In both Wt and Dmd mdx myotubes, preincubation with P2RX7 antagonist A438079 (100 μM), 3-MA (5 mM) or GA (75 nM) produced significant reductions in eATP-induced EtBr fluorescence. Three-MA alone blocked EtBr background signal in unstimulated cells. ( C ) Summary data of differences in LDH release from Wt, Dmd mdx and Dmd mdx p2rx7 −/− myoblasts following 30 min exposure to 3 mM eATP, and Wt and Dmd mdx in the absence or presence of autophagy and P2RX7 inhibitors (3-MA [5 mM] and A804598 [100 nM], respectively). ( D ) Representative western blots showing P2RX7 and LC3-II levels in Wt, Dmd mdx and Dmd mdx p2rx7 −/− double-mutant myotubes incubated for 30 min with 1 mM BzATP. Graph depicts fold change in LC3-II levels in Dmd mdx and Dmd mdx p2rx7 −/− myotubes compared to Wt myotubes following 30 min exposure to 1 mM BzATP. Means +/− SE, n = 5, P

    Techniques Used: Fluorescence, Produced, Western Blot, Mutagenesis, Incubation

    12) Product Images from "G-CSF does not influence C2C12 myogenesis despite receptor expression in healthy and dystrophic skeletal muscle"

    Article Title: G-CSF does not influence C2C12 myogenesis despite receptor expression in healthy and dystrophic skeletal muscle

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2014.00170

    Identification of G-CSFR in rodent and human skeletal muscle. (A) cDNA fragment amplified during Real Time-PCR using the primers described in Table 1 , separated on a 1.8% Sybr safe (Invitrogen) agarose gel and exposed to UV light. (B) Western blot image identifying G-CSFR in rodent and human skeletal muscle in vitro and ex vivo . (C) Western blot for G-CSFR in positive (BAF/3[G]) and negative (293T) control cells. (D) G-CSFR after deglycosylation with PGNase. C 2 B (C2C12 myoblasts), C 2 T (C2C12 myotubes), Dia (diaphragm muscle from C57BL/10 mice), TA (tibialis anterior muscle from C57BL/10 mice), H p B (human primary myoblasts), H p T (human primary myotubes) H VL (human vastus lateralis muscle), BAF/3[G] (murine pro B cell line overexpressing G-CSFR) and 293T (human embryonic kidney 293T cell line). Experiments were performed in triplicate.
    Figure Legend Snippet: Identification of G-CSFR in rodent and human skeletal muscle. (A) cDNA fragment amplified during Real Time-PCR using the primers described in Table 1 , separated on a 1.8% Sybr safe (Invitrogen) agarose gel and exposed to UV light. (B) Western blot image identifying G-CSFR in rodent and human skeletal muscle in vitro and ex vivo . (C) Western blot for G-CSFR in positive (BAF/3[G]) and negative (293T) control cells. (D) G-CSFR after deglycosylation with PGNase. C 2 B (C2C12 myoblasts), C 2 T (C2C12 myotubes), Dia (diaphragm muscle from C57BL/10 mice), TA (tibialis anterior muscle from C57BL/10 mice), H p B (human primary myoblasts), H p T (human primary myotubes) H VL (human vastus lateralis muscle), BAF/3[G] (murine pro B cell line overexpressing G-CSFR) and 293T (human embryonic kidney 293T cell line). Experiments were performed in triplicate.

    Techniques Used: Amplification, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Western Blot, In Vitro, Ex Vivo, Mouse Assay

    13) Product Images from "Adiponectin Increases Skeletal Muscle Mitochondrial Biogenesis by Suppressing Mitogen-Activated Protein Kinase Phosphatase-1"

    Article Title: Adiponectin Increases Skeletal Muscle Mitochondrial Biogenesis by Suppressing Mitogen-Activated Protein Kinase Phosphatase-1

    Journal: Diabetes

    doi: 10.2337/db11-1475

    Adiponectin induces p38 MAPK phosphorylation and PGC-1α expression but decreases MKP1 protein levels in mouse skeletal muscle and C2C12 myotubes. A : Protein samples were prepared from the gastrocnemius muscle of WT and Adipoq −/− mice. For adiponectin reconstitution, Ad-Acrp30 was injected into indicated mice through the tail vein. Ad-GFP was used as control treatment. Tissues were collected 3 days after injection with overnight fasting. Relative protein levels of phospho-p38 (p-p38) MAPK, p38 MAPK, and MKP1 were measured by Western blot; n = 8. B : Fully differentiated C2C12 myotubes were cocultured overnight with FAO cells transduced with Ad-Acrp30 or Ad-GFP (control [Con]) in DMEM without serum. Protein levels were quantified by Western blot using indicated antibodies; n = 6. * P
    Figure Legend Snippet: Adiponectin induces p38 MAPK phosphorylation and PGC-1α expression but decreases MKP1 protein levels in mouse skeletal muscle and C2C12 myotubes. A : Protein samples were prepared from the gastrocnemius muscle of WT and Adipoq −/− mice. For adiponectin reconstitution, Ad-Acrp30 was injected into indicated mice through the tail vein. Ad-GFP was used as control treatment. Tissues were collected 3 days after injection with overnight fasting. Relative protein levels of phospho-p38 (p-p38) MAPK, p38 MAPK, and MKP1 were measured by Western blot; n = 8. B : Fully differentiated C2C12 myotubes were cocultured overnight with FAO cells transduced with Ad-Acrp30 or Ad-GFP (control [Con]) in DMEM without serum. Protein levels were quantified by Western blot using indicated antibodies; n = 6. * P

    Techniques Used: Pyrolysis Gas Chromatography, Expressing, Mouse Assay, Injection, Western Blot, Transduction

    Overexpression of MKP1 attenuates adiponectin-enhanced mitochondrial biogenesis in C2C12 myotubes. MKP1 was overexpressed in differentiated C2C12 myotubes using an adenovirus vector-encoding mouse MKP1 ( A – C and E ). Control cells were transduced with viral vector encoding GFP or LacZ, which was used to avoid interference with fluorescent readings. Twenty-four hours after adenovirus transduction, some cells were treated with adiponectin overnight using the coculture system in which adiponectin was secreted from Ad-Acrp30–transduced FAO cells ( E ). Protein levels of phospho-p38 (p-p38) MAPK, PGC-1α, and MKP1 were detected using Western blot ( A ). Mitochondrial content was measured using MitoTracker Green ( B and E ). Citrate synthase activity was measured using cell lysates ( C ). For A – C and E , n = 6, * P
    Figure Legend Snippet: Overexpression of MKP1 attenuates adiponectin-enhanced mitochondrial biogenesis in C2C12 myotubes. MKP1 was overexpressed in differentiated C2C12 myotubes using an adenovirus vector-encoding mouse MKP1 ( A – C and E ). Control cells were transduced with viral vector encoding GFP or LacZ, which was used to avoid interference with fluorescent readings. Twenty-four hours after adenovirus transduction, some cells were treated with adiponectin overnight using the coculture system in which adiponectin was secreted from Ad-Acrp30–transduced FAO cells ( E ). Protein levels of phospho-p38 (p-p38) MAPK, PGC-1α, and MKP1 were detected using Western blot ( A ). Mitochondrial content was measured using MitoTracker Green ( B and E ). Citrate synthase activity was measured using cell lysates ( C ). For A – C and E , n = 6, * P

    Techniques Used: Over Expression, Plasmid Preparation, Transduction, Pyrolysis Gas Chromatography, Western Blot, Activity Assay

    14) Product Images from "Calpain-1 is required for hydrogen peroxide-induced myotube atrophy"

    Article Title: Calpain-1 is required for hydrogen peroxide-induced myotube atrophy

    Journal:

    doi: 10.1152/ajpcell.00497.2008

    Cysteine protease requirement for H2 O2 -induced myotube atrophy.
    Figure Legend Snippet: Cysteine protease requirement for H2 O2 -induced myotube atrophy.

    Techniques Used:

    Calpain-1 interference inhibits H 2 O 2 -induced protease activity. Fully differentiated C2C12 myotubes were transfected with control (mismatched) or calpain-1 siRNA and subsequently treated with either 0 μM vehicle (control) or 25 μM H 2 O
    Figure Legend Snippet: Calpain-1 interference inhibits H 2 O 2 -induced protease activity. Fully differentiated C2C12 myotubes were transfected with control (mismatched) or calpain-1 siRNA and subsequently treated with either 0 μM vehicle (control) or 25 μM H 2 O

    Techniques Used: Activity Assay, Transfection

    Protease activity in H 2 O 2 induced atrophying myotubes. A : αII-spectrin protease substrate cleavage. αII-spectrin fragments are representative of the calpain-generated 150- and 145-kDa fragments. Fully differentiated C2C12 myotubes were
    Figure Legend Snippet: Protease activity in H 2 O 2 induced atrophying myotubes. A : αII-spectrin protease substrate cleavage. αII-spectrin fragments are representative of the calpain-generated 150- and 145-kDa fragments. Fully differentiated C2C12 myotubes were

    Techniques Used: Activity Assay, Generated

    Cell viability and oxidative stress in C2C12 myotubes treated with H 2 O 2 . A : cell viability. Fully differentiated myotubes were treated with graded concentrations (0 μM vehicle, 25 μM, 50 μM, 100 μM, or 200 μM final
    Figure Legend Snippet: Cell viability and oxidative stress in C2C12 myotubes treated with H 2 O 2 . A : cell viability. Fully differentiated myotubes were treated with graded concentrations (0 μM vehicle, 25 μM, 50 μM, 100 μM, or 200 μM final

    Techniques Used:

    Calpain-1 interference attenuates myotube atrophy. Fully differentiated C2C12 myotubes were transfected with control (mismatched), calpain-1, calpain-2, or caspase-3 small interfering (si)RNA and subsequently treated with 0 μM vehicle (control),
    Figure Legend Snippet: Calpain-1 interference attenuates myotube atrophy. Fully differentiated C2C12 myotubes were transfected with control (mismatched), calpain-1, calpain-2, or caspase-3 small interfering (si)RNA and subsequently treated with 0 μM vehicle (control),

    Techniques Used: Transfection

    Time course of calpain-1 mRNA expression in H 2 O 2 -treated myotubes. Fully differentiated C2C12 myotubes were transfected with control (mismatched) or calpain-1 siRNA and subsequently treated with 0 μM vehicle (control), or 25 μM H 2 O 2 for
    Figure Legend Snippet: Time course of calpain-1 mRNA expression in H 2 O 2 -treated myotubes. Fully differentiated C2C12 myotubes were transfected with control (mismatched) or calpain-1 siRNA and subsequently treated with 0 μM vehicle (control), or 25 μM H 2 O 2 for

    Techniques Used: Expressing, Transfection

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    ATCC murine c2c12 myotubes
    Unique patterns of accessible chromatin in differentiated murine myotubes during hyperammonemia are identified by ATAC-Seq. A , heat map of all differentially accessible chromatin (DAC) (annotated and nonannotated) from ATAC-Seq analysis of differentiated <t>C2C12</t> myotubes that were either untreated (UnT) or treated with 3 h or 24 h of 10 mM ammonium acetate (Am). B , Venn diagram showing unique and shared DAC in UnT myotubes compared with 3hAm and 24hAm. C , heat map and schematic showing differential peaks that either become significantly more accessible (open) or less accessible (closed) at 3hAm returned to baseline (UnT) at 24 h, termed early transient change cluster. Stacked bar chart showing manually curated canonical pathways that are significantly enriched in the early transient change ATAC-Seq cluster. D , heat map and schematic showing differential peaks that are changed significantly only at 24hAm termed late change cluster. Stacked bar chart showing manually curated canonical pathways that are significantly enriched in the late change ATAC-Seq cluster. E , heat map and schematic showing differential peaks that open (or close) significantly at 3hAm and become more open (or more closed) at 24hAm and termed “persistent” change cluster. Stacked bar chart shows manually curated canonical pathways that are significantly enriched in the “persistent” change cluster. F , Venn diagrams of unique and shared DAC/DEG in “like-clusters,” between ATAC-Seq (A) and RNA-Seq (R). G , Venn diagrams of unique and shared DAC/DEG in “dissimilar-clusters.” H , integration of enriched pathways in both the late change ATAC-Seq DAC and late change RNA-Seq ordered by significance. I , heat map of ATAC-Seq (At), RNA-Seq (R), and proteomics (P) DAC/DEG/DEP expression levels. All cellular experiments were performed in three biological replicates. Significance for differential peaks in the ATAC-Seq dataset was set at p
    Murine C2c12 Myotubes, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC myotube differentiation
    Effects of PYCP on the expression of TNF-R1 in TNF-α-treated C2C12 <t>myotubes.</t> TNF-R1 protein expression levels were detected by western blot analysis. GAPDH was used as an internal standard. Data are presented as the mean ± standard deviation of three independent experiments. *P
    Myotube Differentiation, supplied by ATCC, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC myotubes differentiation
    Palmitic acid treatment of C2C12 myoblasts results in smaller <t>myotubes.</t> Type I ( A ) and Type II ( B ) (green) myotubes were identified by immunofluorescence in differentiated C2C12 myotubes treated with vehicle (Control) or 100 μM PA for 48 h. Nuclei were stained with diamidino-2-phenylindole (DAPI) (blue). Length and diameter of type I ( C , D ) and type II ( E , F ) were measured; ( G ) number of stained myotubes for anti-MyHC1 or anti-MyHC2 were counted per field; ( H ) Fusion index; ( I ) mRNA expression of genes related to glycolysis, fatty acid oxidation, and mitochondria were measured by RT-qPCR. mRNA expression was normalized to ribosomal protein, large P0 ( Rpl0 ) expression and are expressed as relative to control levels * ,# p
    Myotubes Differentiation, supplied by ATCC, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Unique patterns of accessible chromatin in differentiated murine myotubes during hyperammonemia are identified by ATAC-Seq. A , heat map of all differentially accessible chromatin (DAC) (annotated and nonannotated) from ATAC-Seq analysis of differentiated C2C12 myotubes that were either untreated (UnT) or treated with 3 h or 24 h of 10 mM ammonium acetate (Am). B , Venn diagram showing unique and shared DAC in UnT myotubes compared with 3hAm and 24hAm. C , heat map and schematic showing differential peaks that either become significantly more accessible (open) or less accessible (closed) at 3hAm returned to baseline (UnT) at 24 h, termed early transient change cluster. Stacked bar chart showing manually curated canonical pathways that are significantly enriched in the early transient change ATAC-Seq cluster. D , heat map and schematic showing differential peaks that are changed significantly only at 24hAm termed late change cluster. Stacked bar chart showing manually curated canonical pathways that are significantly enriched in the late change ATAC-Seq cluster. E , heat map and schematic showing differential peaks that open (or close) significantly at 3hAm and become more open (or more closed) at 24hAm and termed “persistent” change cluster. Stacked bar chart shows manually curated canonical pathways that are significantly enriched in the “persistent” change cluster. F , Venn diagrams of unique and shared DAC/DEG in “like-clusters,” between ATAC-Seq (A) and RNA-Seq (R). G , Venn diagrams of unique and shared DAC/DEG in “dissimilar-clusters.” H , integration of enriched pathways in both the late change ATAC-Seq DAC and late change RNA-Seq ordered by significance. I , heat map of ATAC-Seq (At), RNA-Seq (R), and proteomics (P) DAC/DEG/DEP expression levels. All cellular experiments were performed in three biological replicates. Significance for differential peaks in the ATAC-Seq dataset was set at p

    Journal: The Journal of Biological Chemistry

    Article Title: Integrated multiomics analysis identifies molecular landscape perturbations during hyperammonemia in skeletal muscle and myotubes

    doi: 10.1016/j.jbc.2021.101023

    Figure Lengend Snippet: Unique patterns of accessible chromatin in differentiated murine myotubes during hyperammonemia are identified by ATAC-Seq. A , heat map of all differentially accessible chromatin (DAC) (annotated and nonannotated) from ATAC-Seq analysis of differentiated C2C12 myotubes that were either untreated (UnT) or treated with 3 h or 24 h of 10 mM ammonium acetate (Am). B , Venn diagram showing unique and shared DAC in UnT myotubes compared with 3hAm and 24hAm. C , heat map and schematic showing differential peaks that either become significantly more accessible (open) or less accessible (closed) at 3hAm returned to baseline (UnT) at 24 h, termed early transient change cluster. Stacked bar chart showing manually curated canonical pathways that are significantly enriched in the early transient change ATAC-Seq cluster. D , heat map and schematic showing differential peaks that are changed significantly only at 24hAm termed late change cluster. Stacked bar chart showing manually curated canonical pathways that are significantly enriched in the late change ATAC-Seq cluster. E , heat map and schematic showing differential peaks that open (or close) significantly at 3hAm and become more open (or more closed) at 24hAm and termed “persistent” change cluster. Stacked bar chart shows manually curated canonical pathways that are significantly enriched in the “persistent” change cluster. F , Venn diagrams of unique and shared DAC/DEG in “like-clusters,” between ATAC-Seq (A) and RNA-Seq (R). G , Venn diagrams of unique and shared DAC/DEG in “dissimilar-clusters.” H , integration of enriched pathways in both the late change ATAC-Seq DAC and late change RNA-Seq ordered by significance. I , heat map of ATAC-Seq (At), RNA-Seq (R), and proteomics (P) DAC/DEG/DEP expression levels. All cellular experiments were performed in three biological replicates. Significance for differential peaks in the ATAC-Seq dataset was set at p

    Article Snippet: In vitro cell culture studies were performed in differentiated murine C2C12 myotubes (American Type Culture Collection CRL 1722; American Type Culture Collection) as described by us previously ( ).

    Techniques: RNA Sequencing Assay, Expressing

    ATAC-Seq footprinting analysis was combined with RNA-Seq upstream regulator analysis to identify patterns of transcription factor accessibility during hyperammonemia. A , Venn diagram of transcription factor–binding sites (motifs) that were differentially accessible on ATAC-Seq footprinting analysis of C2C12 myotubes treated with 3 h or 24 h of 10 mM ammonium acetate (Am) compared with untreated (UnT) or 3hAm-treated myotubes. B , heat maps of differentially accessible transcription factor–binding sites identified on ATAC-Seq footprinting analysis of C2C12 myotubes in the early transient, pseudosilent, late, and persistent change clusters. C , significantly enriched canonical pathways identified in the late and early transient change clusters identified on ATAC-Seq footprinting analyses. D , unfolded protein response transcription factor assay for validation of ATAC-Seq footprinting analysis. E , ATAC-Seq footprinting plot showing decreased accessibility (indicating increased likelihood of binding) for all nuclear respiratory factor-1 (NRF1) motifs at 3hAm versus UnT. F , predicted upstream regulator network and their downstream targets in the cellular RNA-Seq differentially expressed genes (DEG) from the late change cluster. Significance for cellular RNA-Seq DEG was taken at false discovery rate (FDR)

    Journal: The Journal of Biological Chemistry

    Article Title: Integrated multiomics analysis identifies molecular landscape perturbations during hyperammonemia in skeletal muscle and myotubes

    doi: 10.1016/j.jbc.2021.101023

    Figure Lengend Snippet: ATAC-Seq footprinting analysis was combined with RNA-Seq upstream regulator analysis to identify patterns of transcription factor accessibility during hyperammonemia. A , Venn diagram of transcription factor–binding sites (motifs) that were differentially accessible on ATAC-Seq footprinting analysis of C2C12 myotubes treated with 3 h or 24 h of 10 mM ammonium acetate (Am) compared with untreated (UnT) or 3hAm-treated myotubes. B , heat maps of differentially accessible transcription factor–binding sites identified on ATAC-Seq footprinting analysis of C2C12 myotubes in the early transient, pseudosilent, late, and persistent change clusters. C , significantly enriched canonical pathways identified in the late and early transient change clusters identified on ATAC-Seq footprinting analyses. D , unfolded protein response transcription factor assay for validation of ATAC-Seq footprinting analysis. E , ATAC-Seq footprinting plot showing decreased accessibility (indicating increased likelihood of binding) for all nuclear respiratory factor-1 (NRF1) motifs at 3hAm versus UnT. F , predicted upstream regulator network and their downstream targets in the cellular RNA-Seq differentially expressed genes (DEG) from the late change cluster. Significance for cellular RNA-Seq DEG was taken at false discovery rate (FDR)

    Article Snippet: In vitro cell culture studies were performed in differentiated murine C2C12 myotubes (American Type Culture Collection CRL 1722; American Type Culture Collection) as described by us previously ( ).

    Techniques: Footprinting, RNA Sequencing Assay, Binding Assay, Transcription Factor Assay

    Proteomic landscape in differentiated murine myotubes during hyperammonemia. A , heat map of differentially expressed proteins (DEP) in myotubes that were either untreated (UnT) or treated with 3 h or 24 h of 10 mM ammonium acetate (Am). B , Venn diagram of unique DEP in UnT myotubes or those treated with 3hAm and 24hAm and analyzed for differential expression using the following comparisons: 3hAm versus UnT, 24hAm versus 3hAm, and 24hAm versus UnT. C , heat map of myotube DEP with significant increase or decrease in expression at 3hAm with a return to baseline (UnT) expression at 24 h, termed early transient change. Stacked bar chart showing manually curated canonical pathways that are significantly enriched in the early transient change cluster on proteomics. D , heat map of myotube DEP that increase or decrease in expression significantly only at 24hAm versus 3hAm and/or UnT, termed late change cluster. Stacked bar chart showing manually curated canonical pathways that are significantly enriched in the late change proteomics cluster. E , heat map of myotube DEP that increase or decrease in expression significantly at 3hAm and become more/less expressed by 24hAm, termed persistent change. Stacked bar chart shows manually curated canonical pathways that are significantly enriched in the persistent change proteomics cluster. F , calcium response in differentiated myotubes to 10 mM Am compared with UnT. G , representative immunoblots and densitometries for validation of proteomics changes in NLR family, apoptosis inhibitory protein 1 (Naip1), myosin heavy chain (Myh), caveolin-3 (Cav-3), and B-cell lymphoma 2 (Bcl2). H , representative immunoblots and densitometry of cleaved caspase-3 and poly-ADP ribose polymerase-1 in UnT, 3hAm (10 mM), 24hAm (10 mM)-treated and 4-h 4 mM H 2 O 2 -treated differentiated C2C12 myotubes. Cell viability percentage is shown with the aforementioned treatments and also in 4-h 1 mM H 2 O 2 -treated cells and 4-h 8 mM H 2 O 2 -treated cells. I , representative immunoblots and densitometry of hypoxia-inducible factor 1α in myotubes. All cellular experiments were performed in three biological replicates. Significance for DEP in the cellular proteomic datasets was set at p

    Journal: The Journal of Biological Chemistry

    Article Title: Integrated multiomics analysis identifies molecular landscape perturbations during hyperammonemia in skeletal muscle and myotubes

    doi: 10.1016/j.jbc.2021.101023

    Figure Lengend Snippet: Proteomic landscape in differentiated murine myotubes during hyperammonemia. A , heat map of differentially expressed proteins (DEP) in myotubes that were either untreated (UnT) or treated with 3 h or 24 h of 10 mM ammonium acetate (Am). B , Venn diagram of unique DEP in UnT myotubes or those treated with 3hAm and 24hAm and analyzed for differential expression using the following comparisons: 3hAm versus UnT, 24hAm versus 3hAm, and 24hAm versus UnT. C , heat map of myotube DEP with significant increase or decrease in expression at 3hAm with a return to baseline (UnT) expression at 24 h, termed early transient change. Stacked bar chart showing manually curated canonical pathways that are significantly enriched in the early transient change cluster on proteomics. D , heat map of myotube DEP that increase or decrease in expression significantly only at 24hAm versus 3hAm and/or UnT, termed late change cluster. Stacked bar chart showing manually curated canonical pathways that are significantly enriched in the late change proteomics cluster. E , heat map of myotube DEP that increase or decrease in expression significantly at 3hAm and become more/less expressed by 24hAm, termed persistent change. Stacked bar chart shows manually curated canonical pathways that are significantly enriched in the persistent change proteomics cluster. F , calcium response in differentiated myotubes to 10 mM Am compared with UnT. G , representative immunoblots and densitometries for validation of proteomics changes in NLR family, apoptosis inhibitory protein 1 (Naip1), myosin heavy chain (Myh), caveolin-3 (Cav-3), and B-cell lymphoma 2 (Bcl2). H , representative immunoblots and densitometry of cleaved caspase-3 and poly-ADP ribose polymerase-1 in UnT, 3hAm (10 mM), 24hAm (10 mM)-treated and 4-h 4 mM H 2 O 2 -treated differentiated C2C12 myotubes. Cell viability percentage is shown with the aforementioned treatments and also in 4-h 1 mM H 2 O 2 -treated cells and 4-h 8 mM H 2 O 2 -treated cells. I , representative immunoblots and densitometry of hypoxia-inducible factor 1α in myotubes. All cellular experiments were performed in three biological replicates. Significance for DEP in the cellular proteomic datasets was set at p

    Article Snippet: In vitro cell culture studies were performed in differentiated murine C2C12 myotubes (American Type Culture Collection CRL 1722; American Type Culture Collection) as described by us previously ( ).

    Techniques: Expressing, Western Blot

    Mouse muscle RNA-Seq integrated with other datasets. A , heat map of differentially expressed genes (DEG) in the RNA-Seq dataset from gastrocnemius muscle from mice implanted with a miniosmotic pump releasing either ammonium acetate (Am) (2.5 mmol.kg −1 .d −1 ) or PBS for 28 days. Stacked bar chart shows manually curated canonical pathways enriched in the RNA-Seq dataset from mouse muscle tissue. B , heat map of shared late change cluster DEG in myotubes and DEG from hyperammonemic mouse skeletal muscle. C , canonical pathways enriched in RNA-Seq from late change cluster in myotubes and mouse skeletal muscle. D , representative immunoblots and densitometry of cyclin-dependent kinase inhibitor p21 in untreated as well as 3hAm- and 24hAm-treated differentiated C2C12 myotubes. E , Venn diagram of unique and shared DEG between the mouse RNA-Seq dataset and the mouse proteomics dataset. F , correlation matrix of shared DEG/DEP expression in mouse muscle RNA-Seq and mouse muscle proteomics. G , pairwise correlation of shared DEG/DEP expression in mouse muscle RNA-Seq and mouse muscle proteomics. Significance for cellular RNA-Seq DEG was taken at false discovery rate (FDR)

    Journal: The Journal of Biological Chemistry

    Article Title: Integrated multiomics analysis identifies molecular landscape perturbations during hyperammonemia in skeletal muscle and myotubes

    doi: 10.1016/j.jbc.2021.101023

    Figure Lengend Snippet: Mouse muscle RNA-Seq integrated with other datasets. A , heat map of differentially expressed genes (DEG) in the RNA-Seq dataset from gastrocnemius muscle from mice implanted with a miniosmotic pump releasing either ammonium acetate (Am) (2.5 mmol.kg −1 .d −1 ) or PBS for 28 days. Stacked bar chart shows manually curated canonical pathways enriched in the RNA-Seq dataset from mouse muscle tissue. B , heat map of shared late change cluster DEG in myotubes and DEG from hyperammonemic mouse skeletal muscle. C , canonical pathways enriched in RNA-Seq from late change cluster in myotubes and mouse skeletal muscle. D , representative immunoblots and densitometry of cyclin-dependent kinase inhibitor p21 in untreated as well as 3hAm- and 24hAm-treated differentiated C2C12 myotubes. E , Venn diagram of unique and shared DEG between the mouse RNA-Seq dataset and the mouse proteomics dataset. F , correlation matrix of shared DEG/DEP expression in mouse muscle RNA-Seq and mouse muscle proteomics. G , pairwise correlation of shared DEG/DEP expression in mouse muscle RNA-Seq and mouse muscle proteomics. Significance for cellular RNA-Seq DEG was taken at false discovery rate (FDR)

    Article Snippet: In vitro cell culture studies were performed in differentiated murine C2C12 myotubes (American Type Culture Collection CRL 1722; American Type Culture Collection) as described by us previously ( ).

    Techniques: RNA Sequencing Assay, Mouse Assay, Western Blot, Expressing

    Differentially expressed genes in RNA-Seq from differentiated murine myotubes and as compared with myotube proteomics. A , heat map of differentially expressed genes (DEG) from RNA-Seq analysis of C2C12 myotubes that were either untreated (UnT) or treated for 3 h or 24 h with 10 mM ammonium acetate (Am). B , Venn diagram showing unique DEG in differentiated C2C12 myotubes either UnT or treated with Am and analyzed using the following comparisons: 3hAm versus UnT, 24hAm versus 3hAm, and 24hAm treatment versus UnT. C , heat map of myotube DEG that have either increased or decreased expression in 3hAm and return to baseline (UnT) status at 24hAm, termed early transient change cluster. D , heat map of myotube DEG that have either increased or decreased expression at 24hAm versus UnT/3hAm termed late change cluster. Stacked bar chart shows manually curated canonical pathways that are significantly enriched in the late change RNA-Seq cluster. E , heat map of myotube DEG that have either increased (or decreased) expression at 3hAm and whose expression increased (or decreased) further at 24hAm treatment, termed persistent change cluster. Stacked bar chart shows manually curated canonical pathways that are significantly enriched in the persistent change RNA-Seq cluster. F , real-time PCR validation of changes in select components on RNA-Seq that included leucyl-tRNA synthetase (Lars), alpha-1,3-mannosyl-glycoprotein 4-beta- N -acetylglucosaminyltransferase C (Mgat4c), and Bcl2 (B-cell lymphoma 2). G , Venn diagrams showing “like-clusters” and “dissimilar-clusters” of unique and shared genes between myotube RNA-Seq and myotube proteomics. H , canonical pathways enriched in late change RNA-Seq and late change proteomics clusters. All cellular experiments were performed in three biological replicates. Significance for cellular DEG was FDR

    Journal: The Journal of Biological Chemistry

    Article Title: Integrated multiomics analysis identifies molecular landscape perturbations during hyperammonemia in skeletal muscle and myotubes

    doi: 10.1016/j.jbc.2021.101023

    Figure Lengend Snippet: Differentially expressed genes in RNA-Seq from differentiated murine myotubes and as compared with myotube proteomics. A , heat map of differentially expressed genes (DEG) from RNA-Seq analysis of C2C12 myotubes that were either untreated (UnT) or treated for 3 h or 24 h with 10 mM ammonium acetate (Am). B , Venn diagram showing unique DEG in differentiated C2C12 myotubes either UnT or treated with Am and analyzed using the following comparisons: 3hAm versus UnT, 24hAm versus 3hAm, and 24hAm treatment versus UnT. C , heat map of myotube DEG that have either increased or decreased expression in 3hAm and return to baseline (UnT) status at 24hAm, termed early transient change cluster. D , heat map of myotube DEG that have either increased or decreased expression at 24hAm versus UnT/3hAm termed late change cluster. Stacked bar chart shows manually curated canonical pathways that are significantly enriched in the late change RNA-Seq cluster. E , heat map of myotube DEG that have either increased (or decreased) expression at 3hAm and whose expression increased (or decreased) further at 24hAm treatment, termed persistent change cluster. Stacked bar chart shows manually curated canonical pathways that are significantly enriched in the persistent change RNA-Seq cluster. F , real-time PCR validation of changes in select components on RNA-Seq that included leucyl-tRNA synthetase (Lars), alpha-1,3-mannosyl-glycoprotein 4-beta- N -acetylglucosaminyltransferase C (Mgat4c), and Bcl2 (B-cell lymphoma 2). G , Venn diagrams showing “like-clusters” and “dissimilar-clusters” of unique and shared genes between myotube RNA-Seq and myotube proteomics. H , canonical pathways enriched in late change RNA-Seq and late change proteomics clusters. All cellular experiments were performed in three biological replicates. Significance for cellular DEG was FDR

    Article Snippet: In vitro cell culture studies were performed in differentiated murine C2C12 myotubes (American Type Culture Collection CRL 1722; American Type Culture Collection) as described by us previously ( ).

    Techniques: RNA Sequencing Assay, Expressing, Real-time Polymerase Chain Reaction

    Graphical summary. Data were generated from C2C12 myotubes and skeletal muscle from mice treated with 10 mM ammonium acetate (Am) or PBS and from human patients with cirrhosis and controls. Datasets from cells and tissues were created using ATAC-Seq, RNA-Seq, and quantitative proteomics. As shown on the left panels , datasets from C2C12 myotubes were separated into clusters of early transient (increase or decrease in accessibility/expression at 3hAm with a return to baseline [UnT] expression at 24hAm), late (increase or decrease in accessibility/expression at 24hAm versus 3hAm and/or 24hAm versus UnT), persistent (increase or decrease in accessibility/expression at both 3hAm and 24hAm versus UnT), and pseudosilent (increase or decrease in accessibility/expression only at 24hAm versus 3hAm but not with either Am treatment versus UnT) change groups. Footprinting analyses from ATAC-Seq were also parsed into the same clusters. Functional enrichment analyses were performed on myotube dataset clusters and mouse and human datasets. Validation experiments were performed using real-time PCR, immunoblots, flow cytometry, calcium flux, and transcription factor array. Novel processes as targets emerged including postmitotic senescence-like phenotype, calcium signaling, and HIF1α signaling. Am, ammonium acetate; ATAC-Seq, assay for transposase-accessible chromatin with high-throughput sequencing; HIF1α, hypoxia-inducible factor 1-alpha; UnT, untreated.

    Journal: The Journal of Biological Chemistry

    Article Title: Integrated multiomics analysis identifies molecular landscape perturbations during hyperammonemia in skeletal muscle and myotubes

    doi: 10.1016/j.jbc.2021.101023

    Figure Lengend Snippet: Graphical summary. Data were generated from C2C12 myotubes and skeletal muscle from mice treated with 10 mM ammonium acetate (Am) or PBS and from human patients with cirrhosis and controls. Datasets from cells and tissues were created using ATAC-Seq, RNA-Seq, and quantitative proteomics. As shown on the left panels , datasets from C2C12 myotubes were separated into clusters of early transient (increase or decrease in accessibility/expression at 3hAm with a return to baseline [UnT] expression at 24hAm), late (increase or decrease in accessibility/expression at 24hAm versus 3hAm and/or 24hAm versus UnT), persistent (increase or decrease in accessibility/expression at both 3hAm and 24hAm versus UnT), and pseudosilent (increase or decrease in accessibility/expression only at 24hAm versus 3hAm but not with either Am treatment versus UnT) change groups. Footprinting analyses from ATAC-Seq were also parsed into the same clusters. Functional enrichment analyses were performed on myotube dataset clusters and mouse and human datasets. Validation experiments were performed using real-time PCR, immunoblots, flow cytometry, calcium flux, and transcription factor array. Novel processes as targets emerged including postmitotic senescence-like phenotype, calcium signaling, and HIF1α signaling. Am, ammonium acetate; ATAC-Seq, assay for transposase-accessible chromatin with high-throughput sequencing; HIF1α, hypoxia-inducible factor 1-alpha; UnT, untreated.

    Article Snippet: In vitro cell culture studies were performed in differentiated murine C2C12 myotubes (American Type Culture Collection CRL 1722; American Type Culture Collection) as described by us previously ( ).

    Techniques: Generated, Mouse Assay, RNA Sequencing Assay, Expressing, Footprinting, Functional Assay, Real-time Polymerase Chain Reaction, Western Blot, Flow Cytometry, Next-Generation Sequencing

    Effects of PYCP on the expression of TNF-R1 in TNF-α-treated C2C12 myotubes. TNF-R1 protein expression levels were detected by western blot analysis. GAPDH was used as an internal standard. Data are presented as the mean ± standard deviation of three independent experiments. *P

    Journal: Molecular Medicine Reports

    Article Title: Pyropia yezoensis protein protects against TNF-α-induced myotube atrophy in C2C12 myotubes via the NF-κB signaling pathway

    doi: 10.3892/mmr.2021.12125

    Figure Lengend Snippet: Effects of PYCP on the expression of TNF-R1 in TNF-α-treated C2C12 myotubes. TNF-R1 protein expression levels were detected by western blot analysis. GAPDH was used as an internal standard. Data are presented as the mean ± standard deviation of three independent experiments. *P

    Article Snippet: When myoblasts were approximately 80–90% confluent, myotube differentiation was initiated by replacing the growth medium with differentiation medium: DMEM supplemented with 2% FBS.

    Techniques: Expressing, Western Blot, Standard Deviation

    Effects of PYCP on the production of IL-6 in TNF-α-treated C2C12 myotubes. C2C12 myotubes were treated with 20 ng/ml TNF-α and PYCP (25, 50, or 100 µg/ml) for 48 h. IL-6 in the culture media was measured using ELISA. Data are presented as the mean ± standard deviation of three independent experiments. *P

    Journal: Molecular Medicine Reports

    Article Title: Pyropia yezoensis protein protects against TNF-α-induced myotube atrophy in C2C12 myotubes via the NF-κB signaling pathway

    doi: 10.3892/mmr.2021.12125

    Figure Lengend Snippet: Effects of PYCP on the production of IL-6 in TNF-α-treated C2C12 myotubes. C2C12 myotubes were treated with 20 ng/ml TNF-α and PYCP (25, 50, or 100 µg/ml) for 48 h. IL-6 in the culture media was measured using ELISA. Data are presented as the mean ± standard deviation of three independent experiments. *P

    Article Snippet: When myoblasts were approximately 80–90% confluent, myotube differentiation was initiated by replacing the growth medium with differentiation medium: DMEM supplemented with 2% FBS.

    Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation

    Effects of PYCP on the ubiquitin-proteasome system in TNF-α-treated C2C12 myotubes. (A) The atrogin-1/MAFbx and MuRF1 protein expression levels were detected by western blot analysis. GAPDH was used as an internal standard. (B) 20S proteasome activity was assessed by detecting AMC in cell lysates after cleavage from the AMC-tagged peptide LLVY. Data are presented as the mean ± standard deviation of three independent experiments. *P

    Journal: Molecular Medicine Reports

    Article Title: Pyropia yezoensis protein protects against TNF-α-induced myotube atrophy in C2C12 myotubes via the NF-κB signaling pathway

    doi: 10.3892/mmr.2021.12125

    Figure Lengend Snippet: Effects of PYCP on the ubiquitin-proteasome system in TNF-α-treated C2C12 myotubes. (A) The atrogin-1/MAFbx and MuRF1 protein expression levels were detected by western blot analysis. GAPDH was used as an internal standard. (B) 20S proteasome activity was assessed by detecting AMC in cell lysates after cleavage from the AMC-tagged peptide LLVY. Data are presented as the mean ± standard deviation of three independent experiments. *P

    Article Snippet: When myoblasts were approximately 80–90% confluent, myotube differentiation was initiated by replacing the growth medium with differentiation medium: DMEM supplemented with 2% FBS.

    Techniques: Expressing, Western Blot, Activity Assay, Standard Deviation

    Effects of TNF-α and PYCP on the cytotoxicity of C2C12 myotubes. (A) The cell viability of C2C12 myotubes in the presence of 0, 12.5, 25, 50, and 100 µg/ml PYCP for 24 h. (B) The cell viability of C2C12 myotubes treated with 25, 50, and 100 µg/ml PYCP for 24 h and cotreated with 20 ng/ml TNF-α for 24 h.*P

    Journal: Molecular Medicine Reports

    Article Title: Pyropia yezoensis protein protects against TNF-α-induced myotube atrophy in C2C12 myotubes via the NF-κB signaling pathway

    doi: 10.3892/mmr.2021.12125

    Figure Lengend Snippet: Effects of TNF-α and PYCP on the cytotoxicity of C2C12 myotubes. (A) The cell viability of C2C12 myotubes in the presence of 0, 12.5, 25, 50, and 100 µg/ml PYCP for 24 h. (B) The cell viability of C2C12 myotubes treated with 25, 50, and 100 µg/ml PYCP for 24 h and cotreated with 20 ng/ml TNF-α for 24 h.*P

    Article Snippet: When myoblasts were approximately 80–90% confluent, myotube differentiation was initiated by replacing the growth medium with differentiation medium: DMEM supplemented with 2% FBS.

    Techniques:

    Effects of PYCP on the expression of MyoD and myogenin in TNF-α-treated C2C12 myotubes. MyoD and myogenin protein expression levels were detected by western blot analysis. GAPDH was used as an internal standard. Data are presented as the mean ± standard deviation of three independent experiments. *P

    Journal: Molecular Medicine Reports

    Article Title: Pyropia yezoensis protein protects against TNF-α-induced myotube atrophy in C2C12 myotubes via the NF-κB signaling pathway

    doi: 10.3892/mmr.2021.12125

    Figure Lengend Snippet: Effects of PYCP on the expression of MyoD and myogenin in TNF-α-treated C2C12 myotubes. MyoD and myogenin protein expression levels were detected by western blot analysis. GAPDH was used as an internal standard. Data are presented as the mean ± standard deviation of three independent experiments. *P

    Article Snippet: When myoblasts were approximately 80–90% confluent, myotube differentiation was initiated by replacing the growth medium with differentiation medium: DMEM supplemented with 2% FBS.

    Techniques: Expressing, Western Blot, Standard Deviation

    Effects of PYCP on the production of intracellular ROS in TNF-α-treated C2C12 myotubes. C2C12 myotubes were treated with 20 ng/ml TNF-α and PYCP (25, 50, or 100 µg/ml) for 48 h. Intracellular ROS production was measured using DCF-DA and fluorescence intensity analysis. Data are presented as the mean ± standard deviation of three independent experiments. *P

    Journal: Molecular Medicine Reports

    Article Title: Pyropia yezoensis protein protects against TNF-α-induced myotube atrophy in C2C12 myotubes via the NF-κB signaling pathway

    doi: 10.3892/mmr.2021.12125

    Figure Lengend Snippet: Effects of PYCP on the production of intracellular ROS in TNF-α-treated C2C12 myotubes. C2C12 myotubes were treated with 20 ng/ml TNF-α and PYCP (25, 50, or 100 µg/ml) for 48 h. Intracellular ROS production was measured using DCF-DA and fluorescence intensity analysis. Data are presented as the mean ± standard deviation of three independent experiments. *P

    Article Snippet: When myoblasts were approximately 80–90% confluent, myotube differentiation was initiated by replacing the growth medium with differentiation medium: DMEM supplemented with 2% FBS.

    Techniques: Fluorescence, Standard Deviation

    Effects of PYCP on the activation and translocation of NF-κB/p65 in TNF-α-treated C2C12 myotubes. (A) p-IκBα, IκBα, and NF-κB/p65 protein expression levels of cytosolic fractions were detected by western blot analysis. (B) NF-κB/p65 levels were measured in the nuclear fractions by western blot analysis. β-actin and lamin B1 were used as internal controls for the cytosolic and nuclear fractions, respectively. Data are presented as the mean ± standard deviation of three independent experiments. *P

    Journal: Molecular Medicine Reports

    Article Title: Pyropia yezoensis protein protects against TNF-α-induced myotube atrophy in C2C12 myotubes via the NF-κB signaling pathway

    doi: 10.3892/mmr.2021.12125

    Figure Lengend Snippet: Effects of PYCP on the activation and translocation of NF-κB/p65 in TNF-α-treated C2C12 myotubes. (A) p-IκBα, IκBα, and NF-κB/p65 protein expression levels of cytosolic fractions were detected by western blot analysis. (B) NF-κB/p65 levels were measured in the nuclear fractions by western blot analysis. β-actin and lamin B1 were used as internal controls for the cytosolic and nuclear fractions, respectively. Data are presented as the mean ± standard deviation of three independent experiments. *P

    Article Snippet: When myoblasts were approximately 80–90% confluent, myotube differentiation was initiated by replacing the growth medium with differentiation medium: DMEM supplemented with 2% FBS.

    Techniques: Activation Assay, Translocation Assay, Expressing, Western Blot, Standard Deviation

    Effects of PYCP on myotube diameter in TNF-α-treated C2C12 myotubes. Representative images and quantification of myotube diameters are shown for C2C12 myotubes treated with 20 ng/ml TNF-α and PYCP (25, 50, or 100 µg/ml) for 48 h. Images were captured at ×20 magnification (scale bar, 50 µm). Data are presented as the mean ± standard deviation of three independent experiments. *P

    Journal: Molecular Medicine Reports

    Article Title: Pyropia yezoensis protein protects against TNF-α-induced myotube atrophy in C2C12 myotubes via the NF-κB signaling pathway

    doi: 10.3892/mmr.2021.12125

    Figure Lengend Snippet: Effects of PYCP on myotube diameter in TNF-α-treated C2C12 myotubes. Representative images and quantification of myotube diameters are shown for C2C12 myotubes treated with 20 ng/ml TNF-α and PYCP (25, 50, or 100 µg/ml) for 48 h. Images were captured at ×20 magnification (scale bar, 50 µm). Data are presented as the mean ± standard deviation of three independent experiments. *P

    Article Snippet: When myoblasts were approximately 80–90% confluent, myotube differentiation was initiated by replacing the growth medium with differentiation medium: DMEM supplemented with 2% FBS.

    Techniques: Standard Deviation

    Palmitic acid treatment of C2C12 myoblasts results in smaller myotubes. Type I ( A ) and Type II ( B ) (green) myotubes were identified by immunofluorescence in differentiated C2C12 myotubes treated with vehicle (Control) or 100 μM PA for 48 h. Nuclei were stained with diamidino-2-phenylindole (DAPI) (blue). Length and diameter of type I ( C , D ) and type II ( E , F ) were measured; ( G ) number of stained myotubes for anti-MyHC1 or anti-MyHC2 were counted per field; ( H ) Fusion index; ( I ) mRNA expression of genes related to glycolysis, fatty acid oxidation, and mitochondria were measured by RT-qPCR. mRNA expression was normalized to ribosomal protein, large P0 ( Rpl0 ) expression and are expressed as relative to control levels * ,# p

    Journal: International Journal of Molecular Sciences

    Article Title: Palmitic Acid Impairs Myogenesis and Alters Temporal Expression of miR-133a and miR-206 in C2C12 Myoblasts

    doi: 10.3390/ijms22052748

    Figure Lengend Snippet: Palmitic acid treatment of C2C12 myoblasts results in smaller myotubes. Type I ( A ) and Type II ( B ) (green) myotubes were identified by immunofluorescence in differentiated C2C12 myotubes treated with vehicle (Control) or 100 μM PA for 48 h. Nuclei were stained with diamidino-2-phenylindole (DAPI) (blue). Length and diameter of type I ( C , D ) and type II ( E , F ) were measured; ( G ) number of stained myotubes for anti-MyHC1 or anti-MyHC2 were counted per field; ( H ) Fusion index; ( I ) mRNA expression of genes related to glycolysis, fatty acid oxidation, and mitochondria were measured by RT-qPCR. mRNA expression was normalized to ribosomal protein, large P0 ( Rpl0 ) expression and are expressed as relative to control levels * ,# p

    Article Snippet: Cells were seeded in 6-well plates, 100,000 cells per well, and when they reached 100% confluence, they were incubated with DMEM supplemented with 2% horse serum for 5 days to induce myotubes differentiation.

    Techniques: Immunofluorescence, Staining, Expressing, Quantitative RT-PCR

    PA impairs myotube formation and induces a slow-to-fast fiber transition in C2C12 myotubes. Immunofluorescence analysis of MyHC1 ( A ) and MyHC2 ( B ) (green) proteins. C2C12 were grown for five days in differentiation media in the absence (vehicle-control) or in the presence of 100 or 150 μM PA. Nuclei were stained with DAPI (blue). Diameters of type I and type II were measured ( C ). Fusion index ( D ). MyHc1 and MyHC2-positive myotubes counted per field (at least four fields were counted per well) ( E ). Relative expression of metabolic genes by RT-qPCR. mRNA expression was normalized to Rpl0 expression ( F ). Relative expression of genes involved in protein synthesis. Gene expression was normalized to Rpl0 levels and is expressed relative to control ( G ). Representative blots of puromycin incorporation ( H ) and Atrogin-1 ( I ) Western blot. Densitometry of bands were analyzed and normalized by Ponceau staining ( n = 6) ( H ) and GAPDH content ( n = 4) ( I ). * ,# p

    Journal: International Journal of Molecular Sciences

    Article Title: Palmitic Acid Impairs Myogenesis and Alters Temporal Expression of miR-133a and miR-206 in C2C12 Myoblasts

    doi: 10.3390/ijms22052748

    Figure Lengend Snippet: PA impairs myotube formation and induces a slow-to-fast fiber transition in C2C12 myotubes. Immunofluorescence analysis of MyHC1 ( A ) and MyHC2 ( B ) (green) proteins. C2C12 were grown for five days in differentiation media in the absence (vehicle-control) or in the presence of 100 or 150 μM PA. Nuclei were stained with DAPI (blue). Diameters of type I and type II were measured ( C ). Fusion index ( D ). MyHc1 and MyHC2-positive myotubes counted per field (at least four fields were counted per well) ( E ). Relative expression of metabolic genes by RT-qPCR. mRNA expression was normalized to Rpl0 expression ( F ). Relative expression of genes involved in protein synthesis. Gene expression was normalized to Rpl0 levels and is expressed relative to control ( G ). Representative blots of puromycin incorporation ( H ) and Atrogin-1 ( I ) Western blot. Densitometry of bands were analyzed and normalized by Ponceau staining ( n = 6) ( H ) and GAPDH content ( n = 4) ( I ). * ,# p

    Article Snippet: Cells were seeded in 6-well plates, 100,000 cells per well, and when they reached 100% confluence, they were incubated with DMEM supplemented with 2% horse serum for 5 days to induce myotubes differentiation.

    Techniques: Immunofluorescence, Staining, Expressing, Quantitative RT-PCR, Western Blot

    MicroRNA expression in differentiated myotubes treated with vehicle (Control) or 100 µM PA for 48 h. miRNAs expression was measured by Stem-loop RT-qPCR. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Palmitic Acid Impairs Myogenesis and Alters Temporal Expression of miR-133a and miR-206 in C2C12 Myoblasts

    doi: 10.3390/ijms22052748

    Figure Lengend Snippet: MicroRNA expression in differentiated myotubes treated with vehicle (Control) or 100 µM PA for 48 h. miRNAs expression was measured by Stem-loop RT-qPCR. * p

    Article Snippet: Cells were seeded in 6-well plates, 100,000 cells per well, and when they reached 100% confluence, they were incubated with DMEM supplemented with 2% horse serum for 5 days to induce myotubes differentiation.

    Techniques: Expressing, Quantitative RT-PCR