CiteAb

Fig.5.H showing Western Blotting from the publication: Antiapoptotic Protein FAIM2 is targeted by miR-3202, and DUX4 via TRIM21, leading to cell death and defective myogenesis.

Fig.4.F showing Western Blotting from the publication: Antiapoptotic Protein FAIM2 is targeted by miR-3202, and DUX4 via TRIM21, leading to cell death and defective myogenesis.

Fig.3.F showing Western Blotting from the publication: Dickkopf-1 Inhibition Reactivates Wnt/β-Catenin Signaling in Rhabdomyosarcoma, Induces Myogenic Markers In Vitro and Impairs Tumor Cell Survival In Vivo.

Fig.2.E showing Western Blotting from the publication: Dickkopf-1 Inhibition Reactivates Wnt/β-Catenin Signaling in Rhabdomyosarcoma, Induces Myogenic Markers In Vitro and Impairs Tumor Cell Survival In Vivo.

Fig.1.E showing Western Blotting from the publication: Myofiber necroptosis promotes muscle stem cell proliferation via releasing Tenascin-C during regeneration.

Fig.2.H showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Blockade of IGF2R improves muscle regeneration and ameliorates Duchenne muscular dystrophy.

Fig.6.B showing Immunocytochemistry-immunofluorescence from the publication: The Stat3-Fam3a axis promotes muscle stem cell myogenic lineage progression by inducing mitochondrial respiration.

Fig.3.C showing Western Blotting from the publication: Camptothecin exhibits topoisomerase1-independent KMT1A suppression and myogenic differentiation in alveolar rhabdomyosarcoma cells.

Fig.1.A showing Western Blotting from the publication: A specific E3 ligase/deubiquitinase pair modulates TBP protein levels during muscle differentiation.

Fig.5.A showing Western Blotting in a Rattus norvegicus (Rat) sample from the publication: Leucine Promotes Proliferation and Differentiation of Primary Preterm Rat Satellite Cells in Part through mTORC1 Signaling Pathway.

Fig.5.D showing Western Blotting in a Rattus norvegicus (Rat) sample from the publication: Leucine Promotes Proliferation and Differentiation of Primary Preterm Rat Satellite Cells in Part through mTORC1 Signaling Pathway.

Fig.3.F showing Western Blotting from the publication: Insulin and LiCl synergistically rescue myogenic differentiation of FoxO1 over-expressed myoblasts.

Fig.6.C showing Western Blotting from the publication: Epidermal growth factor receptor down-regulation triggers human myoblast differentiation.

Fig.3.A showing Western Blotting from the publication: Epidermal growth factor receptor down-regulation triggers human myoblast differentiation.

Fig.1.C showing Western Blotting from the publication: Epidermal growth factor receptor down-regulation triggers human myoblast differentiation.

Fig.1.B showing Western Blotting from the publication: The homeobox transcription factor Barx2 regulates plasticity of young primary myofibers.

Fig.3.A showing Immunocytochemistry-immunofluorescence from the publication: Inhibition of atrogin-1/MAFbx mediated MyoD proteolysis prevents skeletal muscle atrophy in vivo.

17 images found

Tenascin-C from the tissue microenvironment promotes muscle stem cell maintenance and function through Annexin A2.

In Communications Biology on 5 December 2025 by Cecchini, A., Loreti, M., et al.

Skeletal muscle regeneration occurs through the finely timed activation of resident muscle stem cells (MuSC). Following injury, MuSC exit quiescence, undergo myogenic commitment, and regenerate the muscle. This process is coordinated by tissue microenvironment cues, however the underlying mechanisms regulating MuSC function are still poorly understood. Here, we demonstrate that the extracellular matrix protein Tenascin-C (TnC) promotes MuSC self-renewal and function. Mice lacking TnC exhibit reduced number of MuSC, and defects in MuSC self-renewal, myogenic commitment, and repair. We show that fibro-adipogenic progenitors are the primary cellular source of TnC during regeneration, and that MuSC respond through the surface receptor Annexin A2. We further demonstrate that TnC declines during aging, leading to impaired MuSC function. Aged MuSC exposed to soluble TnC show a rescued ability to both migrate and self-renew in vitro. Overall, our results highlight the pivotal role of TnC during muscle repair in healthy and aging muscle.
© 2025. The Author(s).

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Impaired myogenesis in limb girdle muscular dystrophy type 2B.

In Scientific Reports on 30 September 2025 by Souza, L. S., Ishiba, R., et al.

The skeletal muscle tissue has a remarkable capacity of growth and regeneration. Fusion of myoblasts and myotubes elongation are fundamental processes in muscle development. Previous studies have depicted impaired myogenic processes in animal models and myoblast from human patients with muscle diseases. Here, we evaluated the myogenesis in patients with Limb-girdle Muscle Dystrophy 2B (LGMD2B). Aiming to explain why dysferlin-deficient muscle cells lose its myogenic potential, we used immortalized myoblasts from LGMD2B patients and a cellular DYSF knocking-out model. Myotubes from patients were smaller and containing less myonuclei than control myotubes. Main muscle regulatory factors expression were not altered in these cells. The analysis of the expression of newly described genes associated with muscle fusion and growth, such as MYMK, MYMX, PALMD, SHISA2, COL25A, didn´t show any difference with controls, which is a novel finding. It was also observed that dysferlin deficiency doesn't alter the expression of FAM65B and HDAC6 genes, components of a proposed protein complex that needs to be formed to allow muscle differentiation. Interestingly, morphometric analysis of DYSF knock-out myotubes induced by CRISPR/Cas9 also revealed reduced myogenic capacity with formation of smaller myotubes. These findings suggest that the absence of DYSF itself is sufficient to impair muscle formation in vitro, and that downstream gene and protein expression related to muscle development might depend on the presence and proper function of dysferlin.
© 2025. The Author(s).

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Effect of lactation on postpartum pelvic floor muscle regeneration in preclinical model.

In Npj Women's Health on 16 June 2025 by Boscolo Sesillo, F., Manoochehri, H., et al.

Pelvic floor muscle (PFM) recovery following childbirth is essential for preserving pelvic floor function. Despite this, the impact of parturition and lactation on pelvic muscle stem cells (MuSCs), indispensable for skeletal muscle maintenance and regeneration, remains unknown. We determined that vaginal delivery does not cause mechanical injury of the rat PFMs, enabling us to uncouple the effects of lactation on muscle homeostasis from PFM regeneration following simulated birth injury (SBI). Tibialis anterior (TA) served as non-pelvic control. This novel study demonstrates that in the absence of birth injury, lactation blocks MuSC proliferation in PFM and TA, suggesting that postpartum systemic milieu affects MuSCs in pelvic and non-pelvic muscles. In contrast, SBI negated the inhibitory effect of lactation on MuSCs in PFM but not in TA, indicating that local signals released by the injured muscle overcome systemic inhibitory effects of lactation, which persist in muscles remote from the site of injury.
© The Author(s) 2025.

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Epigenetic control of myogenic identity of human muscle stem cells in Duchenne muscular dystrophy.

In IScience on 20 December 2024 by Massenet, J., Weiss-Gayet, M., et al.

In Duchenne muscular dystrophy (DMD), muscle stem cells' (MuSCs) regenerative capacities are overwhelmed leading to fibrosis. Whether MuSCs have intrinsic defects or are disrupted by their environment is unclear. We investigated cell behavior and gene expression of MuSCs from DMD or healthy human muscles. Proliferation, differentiation, and fusion were unaltered in DMD-MuSCs, but with time, they lost their myogenic identity twice as fast as healthy MuSCs. The rapid drift toward a fibroblast-like cell identity was observed at the clonal level, and resulted from altered expression of epigenetic enzymes. Re-expression of CBX3, SMC3, H2AFV, and H3F3B prevented the MuSC identity drift. Among epigenetic changes, a closing of chromatin at the transcription factor MEF2B locus caused downregulation of its expression and loss of the myogenic fate. Re-expression of MEF2B in DMD-MuSCs restored their myogenic fate. MEF2B is key in the maintenance of myogenic identity in human MuSCs, which is altered in DMD.
© 2024 The Authors.

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StarD7 deficiency switches on glycolysis and promotes mitophagy flux in C2C12 myoblasts.

In The FEBS Journal on 1 January 2024 by Rojas, M. L., Muñoz, J. P., et al.

StarD7 is a member of the START protein family required for phosphatidylcholine delivery to the mitochondria, thus key to maintain mitochondrial structure. Its deficiency has been associated with an impairment of cellular processes, such as proliferation and migration, and it has also been reported that it is needed in myogenic differentiation. Here, we show that StarD7 deficiency in C2C12 muscle cells results in the accumulation of abnormal mitochondria, a reduced number of mitochondria per cell area and increased glycolysis. In addition, StarD7-deficient cells undergo an increase in mitochondria-ER contact sites, reduced connexin 43 expression, and disturbances in lipid handling, evidenced by lipid droplet accumulation and decreased levels in phosphatidylserine synthase 1 and 2 expression. Interestingly, StarD7-deficient cells showed alterations in mitophagy markers. We observed accumulation of LC3B-II and BNIP3 proteins in mitochondria-enriched fractions and accumulation of autophagolysosomal and lysosomal vesicles in StarD7-deficient cells. Furthermore, live-cell imaging experiments of StarD7 knockdown cells expressing mitochondria-targeted mKeima indicated an enhanced mitochondria delivery into lysosomes. Importantly, StarD7 reconstitution in StarD7-deficient cells restores LC3B-II expression in mitochondria-enriched fractions at similar levels to those observed in control cells. Collectively, these findings suggest that StarD7-deficient C2C12 myoblasts are associated with altered cristae structure, disturbances in neutral lipid accumulation, glucose metabolism, and increased mitophagy flux. The alterations mentioned above allow for the maintenance of mitochondrial function.
© 2023 Federation of European Biochemical Societies.

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Antiapoptotic Protein FAIM2 is targeted by miR-3202, and DUX4 via TRIM21, leading to cell death and defective myogenesis.

In Cell Death Dis on 25 April 2022 by Soliman, H. A. N., Toso, E. A., et al.

Fig.5.H

Fig.5.H showing Western Blotting from the publication: Antiapoptotic Protein FAIM2 is targeted by miR-3202, and DUX4 via TRIM21, leading to cell death and defective myogenesis.

shRNA knockdown of FAIM2 promotes myoblast apoptosis and blocks myogenic differentiation.A RTqPCR for FAIM2 expression in LHCN-M2 myoblasts transduced with a lentivector expressing shRNA targeting FAIM2 or empty vector control. Data are presented as mean ± SEM; ****P < 0.0001, by unpaired t-test ... more

shRNA knockdown of FAIM2 promotes myoblast apoptosis and blocks myogenic differentiation.A RTqPCR for FAIM2 expression in LHCN-M2 myoblasts transduced with a lentivector expressing shRNA targeting FAIM2 or empty vector control. Data are presented as mean ± SEM; ****P < 0.0001, by unpaired t-test (n = 3). B Western blot analysis of FAIM2 expression in shRNA knockdown and control LHCN-M2 myoblasts, as in A. C Immunofluorescent staining of TUNEL positive cells (red) and DAPI (blue) in shRNA knockdown (bottom) or control (top) LHCN-M2 myoblasts. D Frequency of TUNEL positive LHCN-M2 cells in shRNA knockdown or control LHCN-M2 myoblasts. Data were taken from Immunofluorescence images of five different wells of each cell line. Data are presented as individual data points plus mean; ****P < 0.0001, by unpaired t-test (n = 5). E Immunofluorescent staining for MHC (red) and DAPI (blue) in FAIM2 knockdown (right) or control (left) LHCN-M2 myoblasts, differentiated for 48 h. F RTqPCR for MYOD1 expression in FAIM2 knockdown and control LHCN-M2 myoblasts during proliferation or after 48 h of differentiation. Data are presented as means ± SEM; ****P < 0.0001 by two-way ANOVA with Tukey’s post hoc test (n = 3). G RTqPCR for MYOGENIN expression in the same cells as in H. Data are presented as means ± SEM; for ns P = 0.1686, ****P < 0.0001 by two-way ANOVA with Tukey’s post hoc test (n = 3). H Western blot analysis of MYOD1 and MYOGENIN expression in FAIM2 knockdown or control LHCN-M2 myoblasts during proliferation or after 24 h of differentiation. I Immunofluorescent staining of MYOD1 (red) and DAPI (blue) in FAIM2 knockdown or control LHCN-M2 myoblasts, differentiated for 48 h. J Immunofluorescent staining of MYOGENIN (red) and DAPI (blue) in FAIM2 knockdown or control LHCN-M2 myoblasts, differentiated for 48 h. less

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Antiapoptotic Protein FAIM2 is targeted by miR-3202, and DUX4 via TRIM21, leading to cell death and defective myogenesis.

In Cell Death Dis on 25 April 2022 by Soliman, H. A. N., Toso, E. A., et al.

Fig.4.F

Fig.4.F showing Western Blotting from the publication: Antiapoptotic Protein FAIM2 is targeted by miR-3202, and DUX4 via TRIM21, leading to cell death and defective myogenesis.

siRNA knockdown of FAIM2 blocks myogenic differentiation.A RTqPCR for FAIM2 expression in LHCN-M2 myoblasts transfected with non-targeting siRNA (siNon-Target) or siRNA targeting FAIM2 (siFAIM2) for 72 h. Data are presented as mean ± SEM; ***P = 0.0001, by unpaired t-test (n = 3). B Western blot ... more

siRNA knockdown of FAIM2 blocks myogenic differentiation.A RTqPCR for FAIM2 expression in LHCN-M2 myoblasts transfected with non-targeting siRNA (siNon-Target) or siRNA targeting FAIM2 (siFAIM2) for 72 h. Data are presented as mean ± SEM; ***P = 0.0001, by unpaired t-test (n = 3). B Western blot for FAIM2 expression in LHCN-M2 myoblasts transfected with non-targeting siRNA (siNon-Target) or siRNA targeting FAIM2 (siFAIM2) for 72 h. C Immunofluorescent staining for MHC (red) and DAPI (blue) of LHCN-M2 myoblasts transfected with non-targeting siRNA (left) or siRNA targeting FAIM2 (right) for 72 h, then cultured for 24 h in differentiation medium. D RTqPCR for MYOD1 expression in LHCN-M2 myoblasts transfected with non-targeting siRNA or siRNA targeting FAIM2 mRNA for 72 h and then differentiated for 24 h. Data are presented as mean ± SEM; for ns P = 0.1463, ****P < 0.0001 by two-way ANOVA with Tukey’s post hoc test (n = 3). E RTqPCR for MYOGENIN expression in the same cells shown in D. Data are presented as means ± SEM; for ns P = 0.9988, **P = 0.0014 by two-way ANOVA with Tukey’s post hoc test (n = 3). F Western blot for MYOD1 and MYOGENIN for the same cells shown in D. G Immunofluorescent staining of MYOGENIN (red) and DAPI (blue) of LHCN-M2iDUX4 myoblasts after transfection with 50 nM FAIM2 siRNA or Non-Target siRNA for 72 h followed by 24 h in differentiation medium. less

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Myofiber necroptosis promotes muscle stem cell proliferation via releasing Tenascin-C during regeneration.

In Cell Res on 1 December 2020 by Zhou, S., Zhang, W., et al.

Fig.1.E

Fig.1.E showing Western Blotting from the publication: Myofiber necroptosis promotes muscle stem cell proliferation via releasing Tenascin-C during regeneration.

Necroptosis-deficient mice exhibit muscle regeneration defects.a Representative of H&E staining of TA muscle cross-sections from both uninjured (Day 0) and injured (7 and 15 days after CTX injection) WT and Mlkl−/− mice. Scale bars, 50 μm. b Quantification of myofiber sizes from cross-sectional a... more

Necroptosis-deficient mice exhibit muscle regeneration defects.a Representative of H&E staining of TA muscle cross-sections from both uninjured (Day 0) and injured (7 and 15 days after CTX injection) WT and Mlkl−/− mice. Scale bars, 50 μm. b Quantification of myofiber sizes from cross-sectional areas (CSAs) of injured mice (7 days after CTX injection) and myofibers with multiple central nuclei out of total cells with central nuclei (in vivo fusion index) 15 days post CTX injection. 3 different views were counted for each mouse. The data are expressed as the means ± SD. n = 6 each for WT and Mlkl−/− mice. c Immunofluorescence staining of MYH3 (green) and Laminin (red) in TA muscle cross sections from both uninjured (Day 0) and injured (4 days after CTX injection) mice. Nuclei were identified by staining with DAPI. Scale bars, 25 μm. d Quantification of the MYH3+ myofiber sizes from CSAs of injured mice (4 days after CTX injection, as representatively shown in c). The sizes of 200 adjacent MYH3+ myofibers were measured for each mouse. Each dot represents an individual myofiber. The data are expressed as the means ± SD. n = 6 each for WT and Mlkl−/− mice. e Immunoblotting analysis of MyoD, MyoG, and MYH3 expression in uninjured (Day 0) or injured (4 days after CTX injection) TA muscles using antibodies as indicated. Whole TA muscle lysates were exacted, as described in Materials and Methods, from 3 mice and pooled together for each condition. HSP70 serves as the loading control. The asterisk (*) denotes the non-specific band. Experiments were repeated independently for three times. f Quantification of the MuSCs in injured TA muscles (3 days after CTX injection) by FACS analysis. MuSCs were isolated as described in Materials and Methods. The histogram represents the percentage of MuSCs (PI−CD11b−CD31−CD45−Sca1−Vcam+ population) out of the total digested mono-nucleus cells. Each dot represents an individual mouse. The data are expressed as the means ± SD. n = 6 each for WT and Mlkl−/− mice. g Immunofluorescence staining of Ki67 (green) and Pax7 (red) in freshly isolated MuSCs (3 days after CTX injection). MuSCs were isolated by FACS and fixed on PDL/Collagen-I pre-coated coverslip, as described in Materials and Methods, followed by immunofluorescence staining using the antibodies as indicated. Nuclei were identified by staining with DAPI. Scale bars, 5 μm. h Quantification of the Ki67+/Pax7+ cells as shown in g. MuSCs isolated from 3 mice were pooled together for immunofluorescence staining in each group. The histogram represents the percentage of Ki67+/Pax7+ cells out of 200 Pax7+cells per genotype. The data are expressed as the means ± SD of 6 images. i qRT-PCR analysis of Ccnd1 mRNA level in MuSCs isolated from injured mice (3 days after CTX injection). The mRNA level of Gapdh was used as the internal control. MuSCs isolated from 3 mice were pooled together for qRT-PCR analysis. The data are expressed as the means ± SD of 3 technical repeats. P values for b, d, and f were determined by unpaired two-tailed t-test; P values for h and i were determined by unpaired two-tailed t-test with Welch’s correction. **P < 0.01; ***P < 0.005. CTX, cardiotoxin; TA, tibialis anterior. less

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Blockade of IGF2R improves muscle regeneration and ameliorates Duchenne muscular dystrophy.

In EMBO Mol Med on 9 January 2020 by Bella, P., Farini, A., et al.

Fig.2.H

Fig.2.H showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Blockade of IGF2R improves muscle regeneration and ameliorates Duchenne muscular dystrophy.

IGF2R binding to CD20 is relevant for myogenic differentiationA–CBioinformatic prediction of IGF2R and CD20 cross‐reactivity. Cartoon representation of the interaction of IGF2 (yellow) and IGF2R domain 11 complex from X‐ray structure (PDB code 2v5p). IGF2R domain 11 AB, CD, EF and HI loops and re... more

IGF2R binding to CD20 is relevant for myogenic differentiationA–CBioinformatic prediction of IGF2R and CD20 cross‐reactivity. Cartoon representation of the interaction of IGF2 (yellow) and IGF2R domain 11 complex from X‐ray structure (PDB code 2v5p). IGF2R domain 11 AB, CD, EF and HI loops and residues (shown in sticks format) involved in the hydrophobic interactions are shown (A). Cartoon representation of the IGF2R domain 11–CD20 (lime) complex obtained from docking simulations and residues involved in hydrophobic interactions is shown in stick format (B). The structure of IGF2R (in red) and IGF2 (green) is shown compared to docking poses of epitope 3. These data show that the helical region (residues 178–184) of the epitope mediates binding to the receptor as well as IGF2. The C‐terminal helix of epitope 3 partially overlaps the first α‐helix of IGF2. ClusPro‐dock IGF2R‐CD20 binding epitope poses corresponding to PDB codes 2v5p‐2oslP, 2v5p‐2oslQ and 2v5p‐3pp4P are coloured in yellow, blue and cyan, respectively (C).DOver‐expressing CD20 in C2C12 myoblasts that co‐expressed CD20 (in green) and IGF2R (in red). DAPI‐labelled nuclei are shown in blue. Scale bars = 75 μm.ERepresentative CD20 and IGF2R immunoprecipitation products immunoblotted for IGF2R and CD20, respectively, in untreated and shCD20‐treated C2C12 cell membranes and whole lysates of proteins. The immunoprecipitation output is shown as IP neg.FMyotube immunofluorescence of cells in proliferation medium (P.M.) and after 2, 4 and 6 days of myogenic differentiation in serum‐free medium. Control (untreated), shCD20‐treated C2C12 cells, C2C12 and HSkM myoblasts pre‐treated with anti‐IGF2R for 24 h were stained. Desmin‐positive myotubes are shown in green. Scale bars = 75 μm.GFusion index quantification after 6 days of differentiation. One‐way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Each experiment was performed in triplicate wells. All values are expressed as mean ± SEM.HRepresentative WB of anti‐myogenin, anti‐Myf5, anti‐MyHC and anti‐β‐actin in total protein lysates obtained from untreated, shCD20‐treated C2C12 cells, and C2C12 and HSkM myoblasts pre‐treated with anti‐IGF2R for 24 h; cells were collected under P.M. and after 2, 4 and 6 days of myogenic differentiation. Densitometric analysis of WB data expressed as the ratio of the indicated antibody/β‐actin in arbitrary units. Two‐way ANOVA test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 in comparison with the results obtained in untreated cells at the corresponding time point. ##P < 0.01; ###P < 0.001; ####P < 0.0001 in comparison with the results obtained in shCD20‐treated C2C12 cells at the corresponding time point. Each experiment was performed in triplicate wells. All values are expressed as the mean ± SEM.Source data are available online for this figure. less

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The Stat3-Fam3a axis promotes muscle stem cell myogenic lineage progression by inducing mitochondrial respiration.

In Nat Commun on 17 April 2019 by Sala, D., Cunningham, T. J., et al.

Fig.6.B

Fig.6.B showing Immunocytochemistry-immunofluorescence from the publication: The Stat3-Fam3a axis promotes muscle stem cell myogenic lineage progression by inducing mitochondrial respiration.

Fam3a rescues impaired commitment of Stat3 knockout (KO) muscle stem cells (MuSCs) in vitro and in vivo. a Immunofluorescence analysis and quantification of myogenin expression in MuSCs infected with shCt or shFam3a coding lentiviruses and cultured in growth conditions for 72 h in the presence or... more

Fam3a rescues impaired commitment of Stat3 knockout (KO) muscle stem cells (MuSCs) in vitro and in vivo. a Immunofluorescence analysis and quantification of myogenin expression in MuSCs infected with shCt or shFam3a coding lentiviruses and cultured in growth conditions for 72 h in the presence or absence of recombinant Fam3a (1000 ng/ml) (n = 3 independent experiments). Scale bar 25 μm. b Immunofluorescence analysis and quantification of myogenin expression in control and Stat3 KO MuSCs cultured in growth conditions for 72 h in the presence or absence of recombinant Fam3a (1000 ng/ml) (n = 5 independent experiments). Scale bar 25 μm. c–e Measurement of the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) of Stat3 KO MuSCs cultured in growth conditions for 3 days in the presence or absence of recombinant Fam3a (1000 ng/ml) (n = 5 independent experiments). The Cell Mito Stress Test was performed using the Seahorse XFp Analyzer. f Scheme of the experimental design for the intramuscular Fam3a treatment in vivo. g, h Immunofluorescence analysis and quantification of Pax7- and myogenin-positive cells in transversal sections of the tibialis anterior muscles of Pax7-CreERWT;Stat3f/f and Pax7-CreER;Stat3f/f male mice 6 days after barium chloride injury. Vehicle or 1 μg of Fam3a recombinant protein was injected intramuscularly 1 day after inducing injury (n = 3–4 animals). Scale bar 25 μm. Data are represented as mean ± SEM (Student’s t test or two-way analysis of variance; *p < 0.05, **p < 0.01, ****p < 0.0001) less

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A specific E3 ligase/deubiquitinase pair modulates TBP protein levels during muscle differentiation.

In Elife on 22 September 2015 by Li, L., Martinez, S. S., et al.

Fig.1.A

Fig.1.A showing Western Blotting from the publication: A specific E3 ligase/deubiquitinase pair modulates TBP protein levels during muscle differentiation.

The ubiquitin-proteasome system regulates TBP protein levels.(A) Treatment of differentiated C2C12 myotubes with the proteasome inhibitor MG132 rescues TBP protein levels. Western blot analysis of cytoplasmic and nuclear extracts from C2C12 myoblasts and myotubes was done using antibodies against... more

The ubiquitin-proteasome system regulates TBP protein levels.(A) Treatment of differentiated C2C12 myotubes with the proteasome inhibitor MG132 rescues TBP protein levels. Western blot analysis of cytoplasmic and nuclear extracts from C2C12 myoblasts and myotubes was done using antibodies against TBP and myogenin. Loading control was done by Ponceau S staining. Myotubes were treated as indicated on differentiation day 6. MG132 was used at a final concentration of 10 μM for 4 hr *, a modified version of myogenin was detected upon MG132 treatment. (B) TBP is ubiquitinated in vivo. 293T cells were transfected with indicated plasmids. Ubiquitin conjugates were purified using Ni-NTA resin under denatured conditions from MG132 treated cells, then subjected to western blot analysis using anti-TBP antibody. (C) TBP can be ubiquitinated by both C2C12 myoblast and myotube whole cell extracts in vitro. In vitro transcribed and translated HA-TBP was incubated with whole cell extracts from C2C12 myoblasts or myotubes for in vitro ubiquitination assays. After the reaction, immunopurified HA-TBP (purified using an antibody against HA-tag) was analyzed by western blot using an antibody against ubiquitin.DOI:http://dx.doi.org/10.7554/eLife.08536.003Protein levels of TBP and TAF4 but not RNA polymerase II significantly decrease during C2C12 differentiation.Western blot analysis of whole-cell extracts prepared from C2C12 myoblasts (D0) and myotubes collected on day 3 (D3) and day 6 (D6) post-differentiation was done using antibodies against TAF4, TBP, myogenin and the carboxy terminal domain (CTD) of the large RNA polymerase II (Pol II) subunit RPB1. Signals in Ponceau S staining were used as loading controls.DOI:http://dx.doi.org/10.7554/eLife.08536.004 less

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Leucine Promotes Proliferation and Differentiation of Primary Preterm Rat Satellite Cells in Part through mTORC1 Signaling Pathway.

In Nutrients on 8 May 2015 by Dai, J. M., Yu, M. X., et al.

Fig.5.A

Fig.5.A showing Western Blotting in a Rattus norvegicus (Rat) sample from the publication: Leucine Promotes Proliferation and Differentiation of Primary Preterm Rat Satellite Cells in Part through mTORC1 Signaling Pathway.

Involvement of mTORC1 in leucine-stimulated differentiation of primary satellite cells. (A) Primary preterm rat satellite cells reaching approximately 80% confluence, were induced to differentiate by differentiation medium. Cells lyse every 24 h and the lysates subjected to Western analysis. MyoD... more

Involvement of mTORC1 in leucine-stimulated differentiation of primary satellite cells. (A) Primary preterm rat satellite cells reaching approximately 80% confluence, were induced to differentiate by differentiation medium. Cells lyse every 24 h and the lysates subjected to Western analysis. MyoD and myogenin densitometry values were adjusted to GAPDH intensity, and then normalized to the control group (d0). *p < 0.05, **p < 0.01 vs. control; (B) Confluent primary satellite cells cultured in differentiation medium with varying concentrations of leucine for 1 h. When indicated by “+”, cells received 50 nM rapamycin. We used western blot assay to detect the expression of mTOR and phospho-mTOR. Phospho-mTOR densitometry values were adjusted to total mTOR intensity, and then normalized to expression from the control group (0 mM leucine); (C) Confluent primary satellite cells were cultured in differentiation medium with different concentrations of leucine for 8 h, followed by western blot analysis. MyoD densitometry values were adjusted to GAPDH intensity and then normalized to expression from the control group (0 mM leucine); (D) Confluent primary satellite cells were cultured in differentiation medium with different concentrations of leucine for 3 days, followed by Western analysis. Myogenin densitometry values were adjusted to GAPDH intensity and then normalized to expression from the control group (0 mM leucine). *p < 0.05, **p < 0.01, ***p < 0.01 vs. control (0 mM leucine). #p < 0.05, ##p < 0.01 vs. 2.0 mM leucine. All data are shown as the mean ± SD of three independent experiments and representative images are shown. less

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Leucine Promotes Proliferation and Differentiation of Primary Preterm Rat Satellite Cells in Part through mTORC1 Signaling Pathway.

In Nutrients on 8 May 2015 by Dai, J. M., Yu, M. X., et al.

Fig.5.D

Fig.5.D showing Western Blotting in a Rattus norvegicus (Rat) sample from the publication: Leucine Promotes Proliferation and Differentiation of Primary Preterm Rat Satellite Cells in Part through mTORC1 Signaling Pathway.

Involvement of mTORC1 in leucine-stimulated differentiation of primary satellite cells. (A) Primary preterm rat satellite cells reaching approximately 80% confluence, were induced to differentiate by differentiation medium. Cells lyse every 24 h and the lysates subjected to Western analysis. MyoD... more

Involvement of mTORC1 in leucine-stimulated differentiation of primary satellite cells. (A) Primary preterm rat satellite cells reaching approximately 80% confluence, were induced to differentiate by differentiation medium. Cells lyse every 24 h and the lysates subjected to Western analysis. MyoD and myogenin densitometry values were adjusted to GAPDH intensity, and then normalized to the control group (d0). *p < 0.05, **p < 0.01 vs. control; (B) Confluent primary satellite cells cultured in differentiation medium with varying concentrations of leucine for 1 h. When indicated by “+”, cells received 50 nM rapamycin. We used western blot assay to detect the expression of mTOR and phospho-mTOR. Phospho-mTOR densitometry values were adjusted to total mTOR intensity, and then normalized to expression from the control group (0 mM leucine); (C) Confluent primary satellite cells were cultured in differentiation medium with different concentrations of leucine for 8 h, followed by western blot analysis. MyoD densitometry values were adjusted to GAPDH intensity and then normalized to expression from the control group (0 mM leucine); (D) Confluent primary satellite cells were cultured in differentiation medium with different concentrations of leucine for 3 days, followed by Western analysis. Myogenin densitometry values were adjusted to GAPDH intensity and then normalized to expression from the control group (0 mM leucine). *p < 0.05, **p < 0.01, ***p < 0.01 vs. control (0 mM leucine). #p < 0.05, ##p < 0.01 vs. 2.0 mM leucine. All data are shown as the mean ± SD of three independent experiments and representative images are shown. less

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Inhibition of atrogin-1/MAFbx mediated MyoD proteolysis prevents skeletal muscle atrophy in vivo.

In PLoS One on 26 March 2009 by Lagirand-Cantaloube, J., Cornille, K., et al.

Fig.3.A

Fig.3.A showing Immunocytochemistry-immunofluorescence from the publication: Inhibition of atrogin-1/MAFbx mediated MyoD proteolysis prevents skeletal muscle atrophy in vivo.

Specific degradation of MyoD during C2C12 myotubes atrophy.A/ Coimmunostaining analysis of MyoD and myogenin in myotubes undergoing atrophy. Myotubes at day 4 of differentiation were treated as described in [7]. Myotubes were fixed and stained with anti-MyoD and anti-myogenin and revealed with go... more

Specific degradation of MyoD during C2C12 myotubes atrophy.A/ Coimmunostaining analysis of MyoD and myogenin in myotubes undergoing atrophy. Myotubes at day 4 of differentiation were treated as described in [7]. Myotubes were fixed and stained with anti-MyoD and anti-myogenin and revealed with goat anti-Rabbit antibody conjugated to Texas Red (c, g, k and l) or with goat anti-mouse antibody conjugated to FITC (d, h, i and p) respectively. DAPI staining shows nuclei (b, f, j and n). Images of a representative field were obtained by indirect immunofluorescence microscopy. (Magnifications are ×400). B/ Proteins were extracted from treated myotubes and subjected to immunoblot analysis with specific anti-MyoD and anti-Tropomyosin antibodies respectively. C/ Depletion of MAFbx by shRNAi prevents MyoD degradation in C2C12 myotubes undergoing atrophy. C2C12 myotubes were transfected by pTER+MAFbx or pTER+control at day 2 of differentiation. Forty-eight hours later myotubes were treated with H202 (225 mM) or were incubated in PBS (starved) and myotubes were harvested 6 h later. Cell lysates were prepared and total proteins were subjected to western blot with anti MyoD, anti MAFbx and anti-α-tubulin antibodies. The graph represents averaged densitometric quantification of the data from three replicate experiments. *P<0.05. D/ Analysis of different cellular proteolysis pathways inhibitors on MyoD protein level during starvation-induced atrophy. C2C12 myotubes at day 4 of differentiation were starved by removal growth medium, amino acids and glucose (starved) for 5 h in the presence of DMS0 (lane 2), H2O (lane 3), 30 µM MG132 (lane 4), 30 µM E64 (lane 5), 50 mM MDL (lane 6) 100 µM chloroquine (lanes 7), 10 mM IC3 (lane 8), 25 mM I2C (lane 9) or refed with fresh medium (lane 10). Cell lysates were prepared 5 h later and 50 µg of total proteins were subjected to western blot with anti–MyoD antibodies (Upper panel). Blot was stripped and reprobed with anti-Troponin T antibodies. E/ Myotubes at day 3 of differentiation were treated with H202 (9 h) and/or were starved by removal of growth medium and incubated in PBS (6 h) in the absence (−) of MG132 or in the presence of 30 µM MG132. (+). Fifty micromgramms of total proteins were subjected to western blot with anti–MyoD and anti-Tropomyosin antibodies. less

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