c2c12 myotubes  (ATCC)

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Fibro‐Adipogenic Progenitors Regulate Orofacial Neuromuscular Junction Regeneration via Myostatin

doi: 10.1002/jcsm.70264

Figure Lengend Snippet: FAPs accounted for the disparity in NMJ regeneration between MAS and TA muscles. (a) AChR and MyHC immunolabelling in myotubes differentiated from MuSCs isolated from MAS or TA muscles. Mature myotubes were treated with or without agrin (100 ng/mL) for 16 h. Scale bar = 50 μm. Graphs in the right panel show the quantifications of differentiation index (upper left), myotube width (upper right), AChR cluster counts normalized to myotube area (lower left), ratio of AChR/myotube area (lower middle) and Chrna1 expression quantified by real‐time quantitative PCR (lower right). n = 3. (b) AChR and MyHC staining of MuSCs single culture and cocultures with different FAPs. Scale bar = 50 μm. Graphs in the lower panel show (from left to right) the quantification of differentiation index, myotube area, AChR cluster density and ratio of AChR/myotube area. n = 3. (c) Schematic of the direct coculture of different FAPs with MAS‐derived MuSCs. (d) Representative images of tdTomato + FAPs and α‐BTX labelled AChRs (green) in recipient MAS and TA muscle. Scale bar = 20 μm. (e) Schematic of the FAPs transplantation assay. (f) Quantification of BTX signals per field. n = 6 mice/group. The data are shown as mean ± SD. Unpaired Student's t test was used in (a, upper right panel). Two‐way ANOVA followed by Tukey's post hoc test was used in (a, lower right panel and f). One‐way ANOVA followed by Tukey's post hoc test was used in (b). ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mature myotubes were treated with 100 ng/mL agrin (HY‐ P79236 , MedChemExpress, China) in DM or conditioned medium (CM) of 7 dpi FAPs supplemented with 2% HS for 16 h. For recombinant MSTN treatment, 100 ng/mL MSTN (HY‐ P72632 , MedChemExpress, China) with or without 500 ng/mL follistatin (HY‐ P70315 , MedChemExpress, China) was administered together with agrin treatment.

Techniques: Muscles, Isolation, Expressing, Real-time Polymerase Chain Reaction, Staining, Derivative Assay, Transplantation Assay

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Fibro‐Adipogenic Progenitors Regulate Orofacial Neuromuscular Junction Regeneration via Myostatin

doi: 10.1002/jcsm.70264

Figure Lengend Snippet: MSTN was the paracrine factor that distinguishes MAS‐derived FAPs from TA‐derived FAPs. (a) AChR and MyHC staining of MuSC single culture or cocultures with conditioned medium (CM) of MAS‐ or TA‐isolated FAPs. Scale bar = 50 μm. (b) Graphs show (from left to right) the quantification of myotube area, average AChR cluster size, AChR cluster counts normalized to myotube area and ratio of AChR/myotube area. n = 6. (c) Venn diagram depicting the overlap among DEGs between 7 dpi MAS‐ and TA‐derived FAPs (orange), NMJ‐regulating genes (green) and genes encoding secreted proteins (blue). (d) Mstn expression levels were quantified by qPCR. n = 3. (e) Myostatin levels were quantified by ELISA in CM from 7 dpi MAS‐ or TA‐derived FAPs. n = 3. The data are shown as mean ± SD. One‐way ANOVA followed by Tukey's post hoc test was used in (b). Unpaired Student's t test was used in (d,e). ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mature myotubes were treated with 100 ng/mL agrin (HY‐ P79236 , MedChemExpress, China) in DM or conditioned medium (CM) of 7 dpi FAPs supplemented with 2% HS for 16 h. For recombinant MSTN treatment, 100 ng/mL MSTN (HY‐ P72632 , MedChemExpress, China) with or without 500 ng/mL follistatin (HY‐ P70315 , MedChemExpress, China) was administered together with agrin treatment.

Techniques: Derivative Assay, Staining, Isolation, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Fibro‐Adipogenic Progenitors Regulate Orofacial Neuromuscular Junction Regeneration via Myostatin

doi: 10.1002/jcsm.70264

Figure Lengend Snippet: Recombinant MSTN suppressed AChR clustering and miR‐206 expression. (a) AChR and MyHC staining of MuSCs cultures treated with or without recombinant MSTN (100 ng/mL) and follistatin (500 ng/mL). Scale bar = 50μm. (b) Graphs show (from left to right) the quantification of differentiation index, myotube area, AChR cluster counts normalized to myotube area and ratio of AChR/myotube area. n = 3. (c) The levels of miR‐206 expression in MuSCs cultures. Mature myotubes differentiated from MAS‐derived MuSCs were treated with control, agrin alone, agrin plus MSTN or agrin plus MSTN and follistatin. (d) AChR and MyHC staining of myotubes transfected with miR‐206 mimics or negative control (NC) prior to treatment with agrin and MSTN. Scale bar = 50 μm. (e) Graphs show (from left to right) the quantification of differentiation index, myotube area, AChR cluster counts normalized to myotube area and ratio of AChR/myotube area. n = 3. The data are shown as mean ± SD. One‐way ANOVA followed by Tukey's post hoc test was used. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mature myotubes were treated with 100 ng/mL agrin (HY‐ P79236 , MedChemExpress, China) in DM or conditioned medium (CM) of 7 dpi FAPs supplemented with 2% HS for 16 h. For recombinant MSTN treatment, 100 ng/mL MSTN (HY‐ P72632 , MedChemExpress, China) with or without 500 ng/mL follistatin (HY‐ P70315 , MedChemExpress, China) was administered together with agrin treatment.

Techniques: Recombinant, Expressing, Staining, Derivative Assay, Control, Transfection, Negative Control

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Fibro‐Adipogenic Progenitors Regulate Orofacial Neuromuscular Junction Regeneration via Myostatin

doi: 10.1002/jcsm.70264

Figure Lengend Snippet: MSTN knockdown reversed the negative effect of MAS FAPs on in vitro AChR clustering. (a) MSTN protein and mRNA level in 7 dpi MAS FAPs transfected with negative control (pGPU6/GFP/Neo‐NC) or knockdown vectors (pGPU6/GFP/Neo‐shMSTN). (b) MSTN protein and mRNA level in 7 dpi TA FAPs transfected with negative control (pcDNA3.1(+)) or overexpression vectors (pcDNA3.1(+)‐MSTN). (c) Representative images of AChR and MyHC staining (left panel). Graphs in the right panel show the quantification of differentiation index (upper left), myotube area (upper right), AChR cluster counts normalized to myotube area (lower left) and ratio of AChR/myotube area (lower right). CM was collected from NC and MSTN KD FAPs and added to myotubes differentiated from MAS‐derived MuSCs. Scale bar = 50 μm. n = 3. (d) Representative images of AChR and MyHC staining (left panel). Graphs in the right panel show the quantification of differentiation index (upper left), myotube area (upper right), AChR cluster density (lower left) and ratio of AChR/myotube area (lower right). CM was collected from NC and MSTN OE FAPs and added to myotubes differentiated from MAS‐derived MuSCs. Scale bar = 50 μm. n = 3. (e,f) Transcript levels of Chrna1 , Chrne , Chrng , Musk and Rapsn in myotubes treated with CM from NC or KD FAPs, and NC or OE FAPs. KD, knock down; OE, overexpression. The data are shown as mean ± SD. Unpaired Student's t test was used in (a–d). Two‐way ANOVA followed by Sidak post hoc test was used in (e,f). ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mature myotubes were treated with 100 ng/mL agrin (HY‐ P79236 , MedChemExpress, China) in DM or conditioned medium (CM) of 7 dpi FAPs supplemented with 2% HS for 16 h. For recombinant MSTN treatment, 100 ng/mL MSTN (HY‐ P72632 , MedChemExpress, China) with or without 500 ng/mL follistatin (HY‐ P70315 , MedChemExpress, China) was administered together with agrin treatment.

Techniques: Knockdown, In Vitro, Transfection, Negative Control, Over Expression, Staining, Derivative Assay

Journal: Biochemistry and Biophysics Reports

Article Title: Effects of branched-chain amino acids on iron deficiency-induced muscle atrophy

doi: 10.1016/j.bbrep.2026.102451

Figure Lengend Snippet: DFO treatment had no toxic or off target effects on C2C12 myotubes. (A) Quantification of total nuclei within myotubes (ANOVA; F (3, 8) = 7.248, p = 0.0114, η 2 = 0.731) (N = 3) (B) Representative analysis images in each group generated by the MyoCount program, used to quantify total nuclei within myotubes. Green, myotubes. Blue dots, nuclei. (C) Representative images of C2C12 myotubes after treatment with DFO, DFO + FeCl 3 , or control. Scale bars, 500 μm. Magnification, 100x. (D) Quantification of OD583nm/1.5 × 10 6 cells (ANOVA; F (2, 6) = 3166, p < 0.001, η 2 = 0.999) (N = 3). Data are presented as the mean ± SD (error bars) from 3 independent experiments, and were analyzed using one-way analysis of variance with eta squared (η 2 ) used to measure effect sizes, followed by a post hoc Tukey–Kramer test. ∗p < 0.05, ∗∗∗p < 0.001. NFI, nuclear fusion index.

Article Snippet: For BCAA stimulation, serum and BCAA were excluded from the medium for 90 min to starve myotubes, then BCAA ( l -Leucine, l -Isoleucine, and l -Valine, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) were added at a concentration of 5 mM ( l -leucine: l -isoleucine: l -valine = 2:1:1) for 45 min or 24 h before harvesting cells for protein extraction [ , ].

Techniques: Generated, Control

Journal: Biochemistry and Biophysics Reports

Article Title: Effects of branched-chain amino acids on iron deficiency-induced muscle atrophy

doi: 10.1016/j.bbrep.2026.102451

Figure Lengend Snippet: BCAA had minimal effects on myotube diameter. (A) Representative immunocytochemical images of myotubes. Green: Myotubes (MyHC staining). Blue: Nuclei (DAPI staining). Scale bars, 100 μm. Magnification, 200x. (B) Quantification of the myotube diameter (N = 3). Data are presented as the mean ± SD (error bars) from 3 independent experiments, and were analyzed using one-way analysis of variance with eta squared (η 2 ) used to measure effect sizes (ANOVA; F (3, 8) = 8.454, p = 0.007, η 2 = 0.760), followed by a post hoc Tukey–Kramer test. ∗p < 0.05, ∗∗∗p < 0.001. (C) The violin plot of the relationships between myotube diameter and four experimental groups. The distribution of data in each group was evaluated. The horizontal axis presents the myotube diameter while the vertical axis presents four experimental groups. (D) Quantification of NFI (N = 3). Data are presented as the mean ± SD (error bars) from 3 independent experiments and were analyzed using one-way analysis of variance with eta squared (η 2 ) used to measure effect sizes (ANOVA; F (3, 8) = 9.374, p = 0.005, η 2 = 0.779), followed by a post hoc Tukey–Kramer test. ∗p < 0.05, ∗∗∗p < 0.001. NFI, nuclear fusion index.

Article Snippet: For BCAA stimulation, serum and BCAA were excluded from the medium for 90 min to starve myotubes, then BCAA ( l -Leucine, l -Isoleucine, and l -Valine, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) were added at a concentration of 5 mM ( l -leucine: l -isoleucine: l -valine = 2:1:1) for 45 min or 24 h before harvesting cells for protein extraction [ , ].

Techniques: Staining

Journal: Biochemistry and Biophysics Reports

Article Title: Effects of branched-chain amino acids on iron deficiency-induced muscle atrophy

doi: 10.1016/j.bbrep.2026.102451

Figure Lengend Snippet: BCAA reduced Atrogin-1 expression in DFO-treated myotubes, but had no effect on MuRF-1 expression. (A) Relative expression of Atrogin-1(F (3, 8) = 46.837, p < 0.001, η 2 = 0.946) (N = 3). (B) Relative expression of MuRF-1(F (3, 8) = 26.29, p < 0.001, η 2 = 0.908) (N = 3). (C) (D) Representative WB blot images showing the protein expression of Atrogin-1 and MuRF-1. Protein was extracted on 24 h after BCAA treatment. Data are presented as the mean ± SD (error bars) from three independent experiments, and were analyzed using one-way analysis of variance (ANOVA) with eta squared (η 2 ) used to measure effect sizes, followed by a post hoc Tukey–Kramer test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: For BCAA stimulation, serum and BCAA were excluded from the medium for 90 min to starve myotubes, then BCAA ( l -Leucine, l -Isoleucine, and l -Valine, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) were added at a concentration of 5 mM ( l -leucine: l -isoleucine: l -valine = 2:1:1) for 45 min or 24 h before harvesting cells for protein extraction [ , ].

Techniques: Expressing

Journal: Biochemistry and Biophysics Reports

Article Title: Effects of branched-chain amino acids on iron deficiency-induced muscle atrophy

doi: 10.1016/j.bbrep.2026.102451

Figure Lengend Snippet: BCAA increased the p-Akt in DFO-treated myotubes after 45 min of BCAA treatment, but had no statistically significant effects on other signaling molecules at the same time point. Representative blots for quantification of (A) p-Akt (F (3, 8) = 6.166, p = 0.018, η 2 = 0.6981), p-mTOR, p-p70S6K, p-4E-BP1, and p-eEF2, (B) p-AMPK, and p-ACC, and (C) p-FOXO1, and p–NF–κB p65. Protein was extracted at 45 min after BCCA treatment. Data are presented as the mean ± SD (error bars) from three independent experiments (N = 3), and were analyzed using one-way analysis of variance (ANOVA) with eta squared (η 2 ) used to measure effect sizes, followed by a post hoc Tukey–Kramer test. ∗p < 0.05.

Article Snippet: For BCAA stimulation, serum and BCAA were excluded from the medium for 90 min to starve myotubes, then BCAA ( l -Leucine, l -Isoleucine, and l -Valine, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) were added at a concentration of 5 mM ( l -leucine: l -isoleucine: l -valine = 2:1:1) for 45 min or 24 h before harvesting cells for protein extraction [ , ].

Techniques:

Journal: Biochemistry and Biophysics Reports

Article Title: Effects of branched-chain amino acids on iron deficiency-induced muscle atrophy

doi: 10.1016/j.bbrep.2026.102451

Figure Lengend Snippet: BCAA increased the phosphorylation of p70S6K in DFO-treated myotubes after 24 h of BCAA treatment, but had no effect on the phosphorylation of AMPK, Akt, or eEF2. (A) Representative blots for quantification of (B) p-AMPK(F (3, 8) = 5.620, p = 0.023, η 2 = 0.678), (C) p-Akt (F (3, 8) = 6.394, p = 0.016, η 2 = 0.706), (D) p-p70S6K(F (3, 8) = 16.43, p < 0.001, η 2 = 0.860), and (E) p-eEF2 (F (3, 8) = 10.23, p = 0.004, η 2 = 0.793). Protein was extracted 24 h after BCCA treatment. Data are presented as the mean ± SD (error bars) from three independent experiments (N = 3), and were analyzed using one-way analysis of variance (ANOVA) with eta squared (η 2 ) used to measure effect sizes, followed by a post hoc Tukey–Kramer test. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: For BCAA stimulation, serum and BCAA were excluded from the medium for 90 min to starve myotubes, then BCAA ( l -Leucine, l -Isoleucine, and l -Valine, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) were added at a concentration of 5 mM ( l -leucine: l -isoleucine: l -valine = 2:1:1) for 45 min or 24 h before harvesting cells for protein extraction [ , ].

Techniques: Phospho-proteomics

Journal: Scientific Reports

Article Title: Noninvasive PET imaging of LPS-induced oxidative stress in skeletal muscle using a ROS-targeting radiotracer

doi: 10.1038/s41598-026-35489-3

Figure Lengend Snippet: [¹⁸F]ROStrace uptake in C2C12 myotubes under oxidative stress conditions. ( a ) Time-dependent uptake of [¹⁸F]ROStrace in LPS-treated C2C12 myotubes. Cells were incubated with the tracer for 10, 30, 60, and 120 min, and intracellular radioactivity was measured using a gamma counter ( n = 5 per time point). Uptake is expressed as the percentage of the injected dose per well (%ID). ( b ) Comparative uptake of [¹⁸F]ROStrace after 60 min of incubation in C2C12 myotubes treated with LPS (0.2 mg/mL), or pretreated with N -acetylcysteine (NAC, 3 mM) or piperlongumine (PL, 10 µM). LPS-treated myotubes showed significantly higher uptake than untreated controls ( p = 0.0005, Cohen’s d = 3.54, 95% CI [− 3.627, − 1.509]), while NAC pretreatment reduced tracer uptake ( p < 0.0001, Cohen’s d = 6.51, 95% CI [1.886, 2.974]). PL pretreatment resulted in significantly elevated uptake compared to untreated controls group ( p = 0.0051, Cohen’s d = 2.54, 95% CI [− 4.707, − 1.217]). Data are presented as mean ± standard deviation (SD); n = 5 for all groups.

Article Snippet: The morphology of C2C12 myoblasts and differentiated myotubes was observed using an optical microscope (CKX53, Olympus, Tokyo, Japan).

Techniques: Incubation, Radioactivity, Injection, Standard Deviation

Journal: Scientific Reports

Article Title: Noninvasive PET imaging of LPS-induced oxidative stress in skeletal muscle using a ROS-targeting radiotracer

doi: 10.1038/s41598-026-35489-3

Figure Lengend Snippet: Biological and structural alterations in LPS-treated C2C12 myotubes. ( a ) Representative phase-contrast images of C2C12 myoblasts, differentiated myotubes, and LPS-treated myotubes (0.2 mg/mL, 24 h). Scale bar = 100 μm. ( b ) Quantification of myotube width in control and LPS-treated groups ( n = 3, p = 0.0183, Cohen’s d = 3.14, 95% CI [1.537, 9.496]). ( c ) Representative fluorescence images of MitoTracker Red CMXRos-stained myoblasts, myotubes, and LPS-treated myotubes. Scale bar = 100 μm. ( d ) Quantification of MitoTracker fluorescence intensity relative to myoblasts ( n = 3; myoblast vs. myotube, p = 0.0003, Cohen’s d = 9.63, 95% CI [− 66.52, − 41.16]; myotube vs. myotube + LPS, p = 0.009, Cohen’s d = 3.87, 95% CI [14.39, 55.00]). ( e ) Relative mRNA expression levels of MuRF-1 ( p = 0.0454, Cohen’s d = 2.34, 95% CI [− 0.8699, − 0.01472]) and Atrogin-1 ( p = 0.0494, Cohen’s d = 2.28, 95% CI [− 0.3060, − 0.0006591]) in control and LPS-treated myotubes assessed by RT-qPCR ( n = 3). Data are presented as mean ± SD.

Article Snippet: The morphology of C2C12 myoblasts and differentiated myotubes was observed using an optical microscope (CKX53, Olympus, Tokyo, Japan).

Techniques: Control, Fluorescence, Staining, Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Noninvasive PET imaging of LPS-induced oxidative stress in skeletal muscle using a ROS-targeting radiotracer

doi: 10.1038/s41598-026-35489-3

Figure Lengend Snippet: Assessment of ROS levels in C2C12 myotubes using DHE fluorescence. ( a ) Representative fluorescence images of C2C12 myotubes stained with dihydroethidium (DHE; 20 µM, 30 min) in control and LPS-treated groups. Scale bar = 100 μm. ( b ) Quantification of DHE fluorescence intensity. LPS-treated myotubes exhibited increased fluorescence compared to controls, although the difference was not statistically significant ( n = 9, p = 0.0079, Cohen’s d = 1.43, 95% CI [− 131.1, − 23.29]). Data are presented as mean ± standard deviation (SD).

Article Snippet: The morphology of C2C12 myoblasts and differentiated myotubes was observed using an optical microscope (CKX53, Olympus, Tokyo, Japan).

Techniques: Fluorescence, Staining, Control, Standard Deviation

Journal: American journal of physiology. Endocrinology and metabolism

Article Title: Hyaluronan 35 prevents endotoxin-mediated dysregulated skeletal muscle proteostasis during ethanol exposure

doi: 10.1152/ajpendo.00283.2025

Figure Lengend Snippet: Studies were done in differentiated C2C12 murine myotubes treated without/with ethanol (EtOH) at/for specified concentrations and times. A-C. Representative immunoblots and densitometry for: A . Puromycin incorporation. B . Phosphorylation of mTOR (p-mTOR Ser2448 ) and downstream signaling target (P70S6K). C . Puromycin incorporation. D . Protein synthesis measured by 3 H phenylalanine incorporation with/without 100mM EtOH for 6h. E . Representative photomicrographs and myotube diameter. Measurements were taken from at least 100 myotubes in each group. F . Cellular membrane stiffness by atomic force microscopy in myotubes with/without 100mM EtOH for 6h. G . Gastrocnemius muscle weights from female pair-fed (PF) and 6%v/v binge mouse model of alcohol-associated liver disease (mALD). (n=4 PF, 6 mALD). H . Contractile responses of quadriceps femoris muscle from C57B/6J pair-fed PF and mALD to different frequencies of stimulation. Both binge and chronic (4-weeks) feeding models were studied. Binge model: N=7 pair-fed (3 male, 4 female), 8 mALD (4 male, 4 female); Chronic model: N=10 in each (4 male/6 female) Myotubes: n=3–4 biological replicates. Data shown as mean±SD. * p<0.05; ** p<0.01; *** p<0.001. Statistical analyses: Panels A-E. One-way ANOVA with Fisher’s least significant difference test. Panel F,G. Student’s t-test for independent groups. Panel H. Contractility studies 2-way mixed effects ANOVA with Fisher’s least significant difference test. To avoid large sample size fallacy, for Panel E, comparisons were made for the average for each replicate in different groups.

Article Snippet: In brief, hiPSC were cultured in mTeSR medium (Stem Cell Technologies, Cambridge, MA) with myogenic induction initiated by sequential culture in skeletal muscle induction medium (AMS Biotechnology, Abingdon, UK), myoblast medium (Amsbio-SKM02) and myotube medium (AMS Biotechnology, Abingdon, UK).

Techniques: Western Blot, Phospho-proteomics, Membrane, Microscopy

Journal: American journal of physiology. Endocrinology and metabolism

Article Title: Hyaluronan 35 prevents endotoxin-mediated dysregulated skeletal muscle proteostasis during ethanol exposure

doi: 10.1152/ajpendo.00283.2025

Figure Lengend Snippet: In vitro studies were done in differentiated C2C12 murine myotubes treated without/with ethanol (EtOH) or lipopolysaccharides (LPS) for specified concentrations for 6h. In vivo studies were done in gastrocnemius muscle from wild-type mice without treatment and in a mouse model of sepsis for 4 or 24h. A,B. Representative immunoblots and densitometries of A. Puromycin incorporation with LPS and B. mTORC1 signaling, including LC3 lipidation in myotubes; C. Gastrocnemius muscle weight; D . Representative immunoblots and densitometry of phosphorylation of P65-NFkB, P70S6K, and RPS6, and LC3 lipidation in gastrocnemius muscle from mice. Panels E-G. Myotube responses to 100mM ethanol, 100ng/mL LPS, and 50mM ethanol and 10ng/mL LPS for 6h. Representative immunoblots and densitometry for E. Puromycin incorporation, F. mTORC1 signaling, including LC3 lipidation. G. Myotube diameter. Statistical analyses for all panels: One-way ANOVA with Fisher’s least significant difference post-hoc testing. Data shown as mean±SD from studies in 3–4 biological replicates in myotubes (Panels A,B,E,F) and gastrocnemius from 3–8 mice. * p<0.05; ** p<0.01; *** p<0.001.

Article Snippet: In brief, hiPSC were cultured in mTeSR medium (Stem Cell Technologies, Cambridge, MA) with myogenic induction initiated by sequential culture in skeletal muscle induction medium (AMS Biotechnology, Abingdon, UK), myoblast medium (Amsbio-SKM02) and myotube medium (AMS Biotechnology, Abingdon, UK).

Techniques: In Vitro, In Vivo, Western Blot, Phospho-proteomics

Journal: American journal of physiology. Endocrinology and metabolism

Article Title: Hyaluronan 35 prevents endotoxin-mediated dysregulated skeletal muscle proteostasis during ethanol exposure

doi: 10.1152/ajpendo.00283.2025

Figure Lengend Snippet: C2C12 myotubes were treated with 100mM ethanol (EtOH) for 6 and 24h, and gastrocnemius muscle was harvested from a mouse model of alcohol-related liver disease (mALD) and pair-fed (PF) controls. Bulk RNAseq and untargeted proteomics were performed, and differentially expressed molecules were identified. A. Dendrograms of hierarchical clustered genes from weighted gene co-expression network analyses (WGCNA) where the y-axis is a dissimilarity index based on topological overlap and the distance between branches representing dissimilarity. Similar branches were grouped into ‘modules,’ signified by each color block below the dendrogram. Box and whisker plots of normalized module Eigengenes from select modules. Table of functional enrichment performed using DAVID Functional Annotation Bioinformatics Microarray Analysis with Kyoto Encyclopedia of Genes and Genomes (KEGG) and REACTOME pathway databases mined. B. Heatmaps of LPS-signaling molecules and HA-metabolism on RNAseq in C2C12 myotubes treated without and with 100mM EtOH. C,D. Real-time PCR quantification of mRNA expression fold change of TLR2, TLR4, and CD44 in ( C ) C2C12 myotubes and ( D ) gastrocnemius muscle from mouse model of alcohol-associated liver disease (mALD) and pair-fed (PF) mice. E-G . Representative immunoblots and densitometry of CD44 expression in ( E ) untreated and ethanol-treated myotubes ( F ) gastrocnemius muscle from PF and mALD mice, and ( G ) phosphorylated P65-NFkB in myotubes without/with TLR4 knockdown. One-way ANOVA with Fisher’s least significant difference post-hoc testing performed. Significance cutoff for differentially expressed molecule was p.adj <0.05 in C2C12 RNAseq and p<0.05 for all other datasets. Myotube data from at least 3 biological replicates and mouse data from 3–4 mice per group. All data expressed as mean±SD. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: In brief, hiPSC were cultured in mTeSR medium (Stem Cell Technologies, Cambridge, MA) with myogenic induction initiated by sequential culture in skeletal muscle induction medium (AMS Biotechnology, Abingdon, UK), myoblast medium (Amsbio-SKM02) and myotube medium (AMS Biotechnology, Abingdon, UK).

Techniques: Biomarker Discovery, Expressing, Blocking Assay, Whisker Assay, Functional Assay, Microarray, Real-time Polymerase Chain Reaction, Western Blot, Knockdown

Journal: American journal of physiology. Endocrinology and metabolism

Article Title: Hyaluronan 35 prevents endotoxin-mediated dysregulated skeletal muscle proteostasis during ethanol exposure

doi: 10.1152/ajpendo.00283.2025

Figure Lengend Snippet: Studies were done in differentiated C2C12 murine or human induced pluripotent stem cell (hiPSC) derived myotubes treated without/with 100mM ethanol, 100ng/mL LPS, or 50mM ethanol and 10ng/mL LPS for 6h without/with 1 mg/ml HA35 . A . Representative immunoblots and densitometry of puromycin incorporation as a measure of protein synthesis in myotubes in response to 100mM ethanol or 100 ng/ml LPS with and without HA35. B . Representative flow cytometry data from myotubes stained with puromycin-Alexa fluor treated without/with HA35, ethanol, and LPS. C . Representative immunoblots and densitometry of mTORC1 signaling and protein homeostasis regulatory molecules (phosphorylation of p65 (p-P65-NFkB Ser536 ), myostatin, and total and phosphorylated P70S6K (p-P70S6K Thr389 ) and ribosomal protein S6 (p-RPS6 Ser240/244 ). D . Mean myotube diameter from differentiated C2C12 myotubes treated with HA35, 100mM ethanol, and 10ng/ml lipopolysaccharide for 6h. E-G. Representative immunoblots and densitometry of ( E ) puromycin incorporation, ( F ) mTORC signaling, including myostatin expression, and ( G ) quantification of mean myotube diameter in myotubes without/with EtOH+LPS without/with HA35. H. Representative immunoblots and densitometry of p-RPS6 Ser240/244 and p-P70S6K Thr389 in hiPSC-derived myotubes. Data expressed as mean±SD from at least 100 myotubes in each group. Data from 3–4 biological replicates expressed as mean±SD. * p<0.05; ** p<0.01; *** p<0.001.

Article Snippet: In brief, hiPSC were cultured in mTeSR medium (Stem Cell Technologies, Cambridge, MA) with myogenic induction initiated by sequential culture in skeletal muscle induction medium (AMS Biotechnology, Abingdon, UK), myoblast medium (Amsbio-SKM02) and myotube medium (AMS Biotechnology, Abingdon, UK).

Techniques: Derivative Assay, Western Blot, Flow Cytometry, Staining, Phospho-proteomics, Expressing