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Santa Cruz Biotechnology mouse anti myod monoclonal antibody

Activation of TRPV1 facilitates myogenesis during the process of muscle regeneration in vivo. A-C Representative western blot and relative protein level of <t>MyoD</t> and myogenin in CTX-induced muscle tissue after treatment with CAP and CPZ at different points in time ( n = 3 animals per experimental group; mean ± SD; Two-way ANOVA). D , F The expression level of MyoD and myogenin mRNA in each group at different points in time ( n = 3 animals per experimental group; mean ± SD; Two-way ANOVA). E Representative immunofluorescence images of MyoD in CTX-induced muscle tissue after treatment with CAP and CPZ at 4d. G-H The relative fluorescence intensity of MyoD and the proportion of MyoD+ /DAPI + double-positive cells in each group ( n = 5 animals per experimental group; mean ± SD; One-way ANOVA). Statistical significance was set at P < 0.05. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Scale bar, 50 μm

Mouse Anti Myod Monoclonal Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1001 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "TRPV1 manipulating polarization of M1/M2 macrophages to promote skeletal muscle regeneration"

Article Title: TRPV1 manipulating polarization of M1/M2 macrophages to promote skeletal muscle regeneration

Journal: Skeletal Muscle

doi: 10.1186/s13395-026-00417-6

Figure Legend Snippet: Activation of TRPV1 facilitates myogenesis during the process of muscle regeneration in vivo. A-C Representative western blot and relative protein level of MyoD and myogenin in CTX-induced muscle tissue after treatment with CAP and CPZ at different points in time ( n = 3 animals per experimental group; mean ± SD; Two-way ANOVA). D , F The expression level of MyoD and myogenin mRNA in each group at different points in time ( n = 3 animals per experimental group; mean ± SD; Two-way ANOVA). E Representative immunofluorescence images of MyoD in CTX-induced muscle tissue after treatment with CAP and CPZ at 4d. G-H The relative fluorescence intensity of MyoD and the proportion of MyoD+ /DAPI + double-positive cells in each group ( n = 5 animals per experimental group; mean ± SD; One-way ANOVA). Statistical significance was set at P < 0.05. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Scale bar, 50 μm

Techniques Used: Activation Assay, In Vivo, Western Blot, Expressing, Immunofluorescence, Fluorescence

The influence of TRPV1 in the C2C12 myoblasts differentiation. A-C The relative fluorescence intensity of TRPV1( n = 5 independent random fields of cells; mean ± SD; One-way ANOVA). D Representative western blot of TRPV1 after treatment with CAP and CPZ in vitro. E The protein expression of TRPV1 ( n = 3 independent replicates in cells; mean ± SD; One-way ANOVA). F Representative western blot of MyoD and myogenin after treatment with CAP and CPZ in differentiation medium at different intervals. G , H The protein expression of MyoD and myogenin among each group ( n = 3 independent replicates in cells; mean ± SD; Two-way ANOVA). I , J The relative fluorescence intensity of MYH3 in multinucleated myotubes in each group time-dependently ( n = 5 independent random fields of cells; mean ± SD; Two-way ANOVA). K Representative Giemsa staining images showed the effect of TRPV1 activation individually on multinucleated myotubes ( n = 5 independent random fields of cells). Statistical significance was set at P < 0.05. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Scale bar, 50 μm, 100 μm

Figure Legend Snippet: The influence of TRPV1 in the C2C12 myoblasts differentiation. A-C The relative fluorescence intensity of TRPV1( n = 5 independent random fields of cells; mean ± SD; One-way ANOVA). D Representative western blot of TRPV1 after treatment with CAP and CPZ in vitro. E The protein expression of TRPV1 ( n = 3 independent replicates in cells; mean ± SD; One-way ANOVA). F Representative western blot of MyoD and myogenin after treatment with CAP and CPZ in differentiation medium at different intervals. G , H The protein expression of MyoD and myogenin among each group ( n = 3 independent replicates in cells; mean ± SD; Two-way ANOVA). I , J The relative fluorescence intensity of MYH3 in multinucleated myotubes in each group time-dependently ( n = 5 independent random fields of cells; mean ± SD; Two-way ANOVA). K Representative Giemsa staining images showed the effect of TRPV1 activation individually on multinucleated myotubes ( n = 5 independent random fields of cells). Statistical significance was set at P < 0.05. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Scale bar, 50 μm, 100 μm

Techniques Used: Fluorescence, Western Blot, In Vitro, Expressing, Staining, Activation Assay

TRPV1 regulates M1/M2 macrophage polarization to promote myogenic differentiation in C2C12 cells. A C2C12 myoblasts were co-cultured with M1 or M2 macrophages for 4 days via a transwell cell culture insert. B Representative Western blot bands of MyoD and MYH3 in C2C12 myoblasts after being co-cultured with M1 or M2 macrophages for 4 days. C , D The protein expression of MyoD and MYH3 in those C2C12 myoblasts which were co-cultured with M1or M2 macrophages after CAP and CPZ treatment ( n = 3 independent replicates in cells; mean ± SD; Two-way ANOVA). E , F Representative immunofluorescence images showed myotubes fusion index in C2C12 cells ( n = 5 independent random fields of cells; mean ± SD; Two-way ANOVA). Statistical significance was set at P < 0.05. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Scale bar, 50 μm

Figure Legend Snippet: TRPV1 regulates M1/M2 macrophage polarization to promote myogenic differentiation in C2C12 cells. A C2C12 myoblasts were co-cultured with M1 or M2 macrophages for 4 days via a transwell cell culture insert. B Representative Western blot bands of MyoD and MYH3 in C2C12 myoblasts after being co-cultured with M1 or M2 macrophages for 4 days. C , D The protein expression of MyoD and MYH3 in those C2C12 myoblasts which were co-cultured with M1or M2 macrophages after CAP and CPZ treatment ( n = 3 independent replicates in cells; mean ± SD; Two-way ANOVA). E , F Representative immunofluorescence images showed myotubes fusion index in C2C12 cells ( n = 5 independent random fields of cells; mean ± SD; Two-way ANOVA). Statistical significance was set at P < 0.05. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Scale bar, 50 μm

Techniques Used: Cell Characterization, Cell Culture, Western Blot, Expressing, Immunofluorescence

Incubation:

Article Title: Characterization of myogenesis in European sea bass ( Dicentrarchus labrax ) using primary white muscle cell cultures
Article Snippet: Then, cells were permeabilized with 0.05% (w/v) saponin (Ref. S-4521, Sigma-Aldrich) in DPBS with 20 mM glycine (Ref. G8898, Sigma-Aldrich) for 8 min. After two washes with DPBS and 20 mM glycine (washing solution) for 5 min each, cells were blocked with 1% (w/v) bovine serum albumin (BSA, Ref. A7030, Sigma-Aldrich) in DPBS with 20 mM glycine for 40 min at room temperature. .. Cells were incubated with mouse monoclonal anti-Pcna (1:100 dilution, Ref. sc-56, Santa Cruz Biotechnology, Heidelberg, Germany), mouse monoclonal anti-Myod (1:100 dilution, Ref. sc-377460, Santa Cruz Biotechnology), or mouse monoclonal anti-Myhc (1:100 dilution; Ref. MF20, Developmental Studies Hybridoma Bank, University of Iowa, USA) for 1 h at 37 °C, followed by two washes with washing solution (5 min each) and incubation with Alexa-546-conjugated goat anti-mouse secondary antibody (1:1500 dilution, Ref. A11003, Thermo Fisher Scientific) for 1 h at 37 °C. .. For nuclei staining, cells were incubated with 0.5 μg/mL DAPI (Ref. d9542, Sigma-Aldrich) for 5 min at room temperature, and for F-actin staining, cells were incubated with Phalloidin CruzFluorTM 488 Conjugate (1:2000 dilution, Ref. sc-363791, Santa Cruz Biotechnology) for 20 min at room temperature.

Article Title: Characterization of myogenesis in European sea bass (Dicentrarchus labrax) using primary white muscle cell cultures.
Article Snippet: Then, cells were permeabilized with 0.05% (w/v) saponin AR TIC LE IN PR ES S (Ref. S-4521, Sigma-Aldrich) in DPBS with 20 mM glycine (Ref. G8898, Sigma-Aldrich) for 8 min. After two washes with DPBS and 20 mM glycine (washing solution) for 5 min each, cells were blocked with 1% (w/v) bovine serum albumin (BSA, Ref. A7030, Sigma-Aldrich) in DPBS with 20 mM glycine for 40 min at room temperature. .. Cells were incubated with mouse monoclonal anti-Pcna (1:100 dilution, Ref. sc-56, Santa Cruz Biotechnology, Heidelberg, Germany), mouse monoclonal anti-Myod (1:100 dilution, Ref. sc-377460, Santa Cruz Biotechnology), or mouse monoclonal anti-Myhc (1:100 dilution; Ref. MF20, Developmental Studies Hybridoma Bank, University of Iowa, USA) for 1 h at 37 oC, followed by two washes with washing solution (5 min each) and incubation with Alexa-546-conjugated goat anti-mouse secondary antibody (1:1500 dilution, Ref. A11003, Thermo Fisher Scientific) for 1 h at 37 oC. .. For nuclei staining, cells were incubated with 0.5 μg/mL DAPI (Ref. d9542, Sigma-Aldrich) for 5 min at room temperature, and for F-actin staining, cells were incubated with Phalloidin CruzFluor 488 Conjugate (1:2000 dilution, Ref. sc-363791, Santa Cruz Biotechnology) for 20 min at room temperature.

Article Title: Duchenne muscular dystrophy progression induced by downhill running is accompanied by increased endomysial fibrosis and oxidative damage DNA in muscle of mdx mice.
Article Snippet: Duchenne muscular dystrophy (DMD) is characterized by progressive muscle necrosis.. One of the major challenges for prescribing physical rehabilitation exercises for DMD patients is associated with the lack of a thorough knowledge of dystrophic muscle responsiveness to exercise.. This study aims to understand the relationship between myogenic regulation, inflammation and oxidative stress parameters, and disease progression induced by downhill running in the skeletal muscle of an experimental model of DMD.

other:

Article Title: PRMT5 mediates FoxO1 methylation and subcellular localization to regulate lipophagy in myogenic progenitors
Article Snippet: Mouse Monoclonal anti-MyoD , Santa Cruz Biotechnology , Cat# sc-377460, RRID: AB_2813984.

Article Title: Lipid droplet dynamics regulate adult muscle stem cell fate
Article Snippet: Mouse monoclonal anti-MyoD , Santa Cruz Biotechnology , Cat# sc-377460, RRID:AB_2813894.

Image Search Results

Journal: Skeletal Muscle

Article Title: TRPV1 manipulating polarization of M1/M2 macrophages to promote skeletal muscle regeneration

doi: 10.1186/s13395-026-00417-6

Figure Lengend Snippet: Activation of TRPV1 facilitates myogenesis during the process of muscle regeneration in vivo. A-C Representative western blot and relative protein level of MyoD and myogenin in CTX-induced muscle tissue after treatment with CAP and CPZ at different points in time ( n = 3 animals per experimental group; mean ± SD; Two-way ANOVA). D , F The expression level of MyoD and myogenin mRNA in each group at different points in time ( n = 3 animals per experimental group; mean ± SD; Two-way ANOVA). E Representative immunofluorescence images of MyoD in CTX-induced muscle tissue after treatment with CAP and CPZ at 4d. G-H The relative fluorescence intensity of MyoD and the proportion of MyoD+ /DAPI + double-positive cells in each group ( n = 5 animals per experimental group; mean ± SD; One-way ANOVA). Statistical significance was set at P < 0.05. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Scale bar, 50 μm

Article Snippet: Deparaffinized sections were blocked with 5% bovine serum albumin (BSA) and incubated overnight at 4 °C with the following primary antibodies: mouse anti-CD86 monoclonal antibody (1:200, Santa Cruz Biotechnology, sc-28347); mouse anti-CD206 polyclonal antibody (1:200, Santa Cruz Biotechnology, sc-58986); rat anti-F4/80 monoclonal antibody (1:100, Abcam, ab1691); rabbit anti-F4/80 monoclonal antibody (1:200, Proteintech, #27044-1-AP); rabbit anti-TRPV1 polyclonal antibody (1:200, ABclonal, A8564); and mouse anti-MyoD monoclonal antibody (1:100, Santa Cruz Biotechnology, sc-377460).

Techniques: Activation Assay, In Vivo, Western Blot, Expressing, Immunofluorescence, Fluorescence

Journal: Skeletal Muscle

Article Title: TRPV1 manipulating polarization of M1/M2 macrophages to promote skeletal muscle regeneration

doi: 10.1186/s13395-026-00417-6

Figure Lengend Snippet: The influence of TRPV1 in the C2C12 myoblasts differentiation. A-C The relative fluorescence intensity of TRPV1( n = 5 independent random fields of cells; mean ± SD; One-way ANOVA). D Representative western blot of TRPV1 after treatment with CAP and CPZ in vitro. E The protein expression of TRPV1 ( n = 3 independent replicates in cells; mean ± SD; One-way ANOVA). F Representative western blot of MyoD and myogenin after treatment with CAP and CPZ in differentiation medium at different intervals. G , H The protein expression of MyoD and myogenin among each group ( n = 3 independent replicates in cells; mean ± SD; Two-way ANOVA). I , J The relative fluorescence intensity of MYH3 in multinucleated myotubes in each group time-dependently ( n = 5 independent random fields of cells; mean ± SD; Two-way ANOVA). K Representative Giemsa staining images showed the effect of TRPV1 activation individually on multinucleated myotubes ( n = 5 independent random fields of cells). Statistical significance was set at P < 0.05. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Scale bar, 50 μm, 100 μm

Techniques: Fluorescence, Western Blot, In Vitro, Expressing, Staining, Activation Assay

Journal: Skeletal Muscle

Article Title: TRPV1 manipulating polarization of M1/M2 macrophages to promote skeletal muscle regeneration

doi: 10.1186/s13395-026-00417-6

Figure Lengend Snippet: TRPV1 regulates M1/M2 macrophage polarization to promote myogenic differentiation in C2C12 cells. A C2C12 myoblasts were co-cultured with M1 or M2 macrophages for 4 days via a transwell cell culture insert. B Representative Western blot bands of MyoD and MYH3 in C2C12 myoblasts after being co-cultured with M1 or M2 macrophages for 4 days. C , D The protein expression of MyoD and MYH3 in those C2C12 myoblasts which were co-cultured with M1or M2 macrophages after CAP and CPZ treatment ( n = 3 independent replicates in cells; mean ± SD; Two-way ANOVA). E , F Representative immunofluorescence images showed myotubes fusion index in C2C12 cells ( n = 5 independent random fields of cells; mean ± SD; Two-way ANOVA). Statistical significance was set at P < 0.05. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Scale bar, 50 μm

Techniques: Cell Characterization, Cell Culture, Western Blot, Expressing, Immunofluorescence

RMS cells are resistant to cell death due to a differentiation phenotype (A and B) Myogenic gene expression was measured by qPCR and western blot analyses in human skeletal muscle myoblasts (hSk) and RH30 cells, n = 3. (C) Abundance of myogenic genes, MYOD1, MYOG, MYH1(MYHC), and TNNT1 from 101 RMS patients retrieved from an R2 Genomics Analysis and Visualization Platform. (D) qPCR analysis comparing expression of MyoD and its respective target genes in myogenic differentiation in RH30-SR cells containing a scrambled-control sh-RNA or sh-RNA targeting MyoD, n = 3. (E) Cells from (D) were treated with TNF or DOX, and cell death was subsequently measured by flow cytometry, n = 3. (F) RH30-SR cells expressing scrambled small guide RNAs (Vector) or a knockdown of MyoD generated by CRISPR/Cas9 deletion (MyoD Δ ), were treated with TNF or DOX, and subsequently measured for cell death, n = 3. (G) GO enrichment analysis for genes with decreased expression in RH30-SR MyoD Δ compared to RH30-SR Vector cells. (H) RH30-SR MyoD Δ cells were reconstituted with control (Vector), the wildtype (WT) form of MyoD, or a mutant form of MyoD (MyoD L122R ). Cells were then exposed to either PBS or TNF for 24 h and cell death was measured and normalized to the untreated RH30-SR MyoD Δ cells. Treatment groups were analyzed with one-way ANOVAs with Tukey’s multiple comparisons n = 3. Data with error bars are depicted as mean ± SEM, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. See also .

Journal: iScience

Article Title: MyoD is essential in rhabdomyosarcoma by promoting survival through differentiation and CYLD

doi: 10.1016/j.isci.2025.113149

Figure Lengend Snippet: RMS cells are resistant to cell death due to a differentiation phenotype (A and B) Myogenic gene expression was measured by qPCR and western blot analyses in human skeletal muscle myoblasts (hSk) and RH30 cells, n = 3. (C) Abundance of myogenic genes, MYOD1, MYOG, MYH1(MYHC), and TNNT1 from 101 RMS patients retrieved from an R2 Genomics Analysis and Visualization Platform. (D) qPCR analysis comparing expression of MyoD and its respective target genes in myogenic differentiation in RH30-SR cells containing a scrambled-control sh-RNA or sh-RNA targeting MyoD, n = 3. (E) Cells from (D) were treated with TNF or DOX, and cell death was subsequently measured by flow cytometry, n = 3. (F) RH30-SR cells expressing scrambled small guide RNAs (Vector) or a knockdown of MyoD generated by CRISPR/Cas9 deletion (MyoD Δ ), were treated with TNF or DOX, and subsequently measured for cell death, n = 3. (G) GO enrichment analysis for genes with decreased expression in RH30-SR MyoD Δ compared to RH30-SR Vector cells. (H) RH30-SR MyoD Δ cells were reconstituted with control (Vector), the wildtype (WT) form of MyoD, or a mutant form of MyoD (MyoD L122R ). Cells were then exposed to either PBS or TNF for 24 h and cell death was measured and normalized to the untreated RH30-SR MyoD Δ cells. Treatment groups were analyzed with one-way ANOVAs with Tukey’s multiple comparisons n = 3. Data with error bars are depicted as mean ± SEM, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. See also .

Article Snippet: Precleared lysates were precipitated with monoclonal mouse anti-human MyoD antibody (Santa Cruz Biotechnology Cat# sc-377460, RRID: AB_2813894 ) or mouse IgG (Millipore Cat# CS200261 ) bound to protein G magnetic beads (saturated with herring sperm DNA and bovine serum albumin).

Techniques: Gene Expression, Western Blot, Expressing, Control, Flow Cytometry, Plasmid Preparation, Knockdown, Generated, CRISPR, Mutagenesis

RMS survival is selectively regulated by MyoD (A) Representative images of immunohistochemistry staining of MyoD and myogenin in patient embryonal and alveolar tumors. (B) Histologic images in (A) were scored for MyoD and myogenin immunohistochemistry staining positivity, n = 5. (C) RH30-SR cells expressing a scrambled-control sh-RNA or sh-RNA targeting myogenin were treated with TNF and DOX for 24 h and subsequently measured for cell death, n = 3. (D) RNA was prepared from cells in (C) and qPCR analysis was performed probing for myogenin and its target genes, n = 3. (E) RH30-SR cells expressing a scrambled-control sh-RNA or sh-RNA against either MyoD, MYF5, myogenin, MRF4, MEF2C, and MEF2D (sh-MRFs) were treated with TNF for 24 h and cell death was subsequently measured, n = 3. (F) CHRONOS Score for MRFs in sarcoma cell lines tested in DepMap. RMS cell lines are highlighted in purple, with a score of < −1 indicating a gene that is essential; ATRT, atypical teratoid rhabdoid tumors; ES, Ewing sarcoma; FS, follicular sarcoma; LMS, leiomyosarcoma; LS, liposarcoma; MRT, malignant rhabdoid tumor; PS, pleomorphic sarcoma; RMS, rhabdomyosarcoma; SS, synovial sarcoma; TS, thyroid sarcoma; US, undifferentiated sarcoma. (G and H) Kaplan-Meier curve, log rank test, showing the correlation of both (G) MyoD and (H) myogenin expression stratified by median patient expression to overall survival of rhabdomyosarcoma patients from an R2 Genomics Analysis and Visualization Platform. (I) CHRONOS Score for p65 in sarcoma cell lines tested in the Cancer Dependency Map. Data with error bars are depicted as mean ± SEM, ∗ p < 0.05; ∗∗ p < 0.01. See also .

Journal: iScience

Article Title: MyoD is essential in rhabdomyosarcoma by promoting survival through differentiation and CYLD

doi: 10.1016/j.isci.2025.113149

Figure Lengend Snippet: RMS survival is selectively regulated by MyoD (A) Representative images of immunohistochemistry staining of MyoD and myogenin in patient embryonal and alveolar tumors. (B) Histologic images in (A) were scored for MyoD and myogenin immunohistochemistry staining positivity, n = 5. (C) RH30-SR cells expressing a scrambled-control sh-RNA or sh-RNA targeting myogenin were treated with TNF and DOX for 24 h and subsequently measured for cell death, n = 3. (D) RNA was prepared from cells in (C) and qPCR analysis was performed probing for myogenin and its target genes, n = 3. (E) RH30-SR cells expressing a scrambled-control sh-RNA or sh-RNA against either MyoD, MYF5, myogenin, MRF4, MEF2C, and MEF2D (sh-MRFs) were treated with TNF for 24 h and cell death was subsequently measured, n = 3. (F) CHRONOS Score for MRFs in sarcoma cell lines tested in DepMap. RMS cell lines are highlighted in purple, with a score of < −1 indicating a gene that is essential; ATRT, atypical teratoid rhabdoid tumors; ES, Ewing sarcoma; FS, follicular sarcoma; LMS, leiomyosarcoma; LS, liposarcoma; MRT, malignant rhabdoid tumor; PS, pleomorphic sarcoma; RMS, rhabdomyosarcoma; SS, synovial sarcoma; TS, thyroid sarcoma; US, undifferentiated sarcoma. (G and H) Kaplan-Meier curve, log rank test, showing the correlation of both (G) MyoD and (H) myogenin expression stratified by median patient expression to overall survival of rhabdomyosarcoma patients from an R2 Genomics Analysis and Visualization Platform. (I) CHRONOS Score for p65 in sarcoma cell lines tested in the Cancer Dependency Map. Data with error bars are depicted as mean ± SEM, ∗ p < 0.05; ∗∗ p < 0.01. See also .

Techniques: Immunohistochemistry, Staining, Expressing, Control

RMS tumors utilize MyoD to resist stress in vivo (A) RH30-SR cells were infected with Vector-control or Yamanaka reprogramming factors (OKMS) and expression of stem cell factors was analyzed by qPCR, n = 3. (B) RH30-SR cells were infected as detailed in (A) and analyzed by qPCR for mesenchymal genes, n = 3. (C) Cells in (A) were treated with TNF for 12 h and cell death was measured as compared to RH30-SR MyoD Δ cells using one-way ANOVA with Dunnett’s multiple comparisons, n = 3. (D) RH30-SR MyoD Δ (clones M29 and M2) and RH30-SR Vector control cells were injected in SCID mice and tumor volumes were measured over time, n = 5. (E) A Kaplan-Meier curve, log rank test, was generated to predict the overall survival of mice with RH30-SR Vector and RH30-SR MyoD Δ tumors, n = 5. (F) RH30-SR Vector and RH30-SR MyoD Δ cells were co-cultured with activated macrophages (0:1, 10:1, and 20:1) and cell survival was subsequently scored by trypan blue exclusion and compared to baseline cell death, analyzed with a two-way ANOVA with Sidak’s multiple comparisons with an interaction of p = 0.0133, n = 3. (G) RH30-SR Vector and RH30-SR MyoD Δ cells were treated with 0 or 10 ng/mL of TNF and 0, 10, or 50 nM of sodium nitroprusside (SNP) and cell survival was subsequently scored and analyzed as in (F), with an interaction of p = 0.0003, n = 3. (H) RH30-SR cells were administered to SCID mice and tumors that formed were subsequently injected with lentiviruses expressing OKMS factors or GFP control. Mice were then treated with 2 doses of vincristine (1.0 mg/kg; days 0 and 7; arrowheads) and tumor volumes were continually measured, n = 5. Data with error bars are depicted as mean ± SEM, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. See also .

Journal: iScience

Article Title: MyoD is essential in rhabdomyosarcoma by promoting survival through differentiation and CYLD

doi: 10.1016/j.isci.2025.113149

Figure Lengend Snippet: RMS tumors utilize MyoD to resist stress in vivo (A) RH30-SR cells were infected with Vector-control or Yamanaka reprogramming factors (OKMS) and expression of stem cell factors was analyzed by qPCR, n = 3. (B) RH30-SR cells were infected as detailed in (A) and analyzed by qPCR for mesenchymal genes, n = 3. (C) Cells in (A) were treated with TNF for 12 h and cell death was measured as compared to RH30-SR MyoD Δ cells using one-way ANOVA with Dunnett’s multiple comparisons, n = 3. (D) RH30-SR MyoD Δ (clones M29 and M2) and RH30-SR Vector control cells were injected in SCID mice and tumor volumes were measured over time, n = 5. (E) A Kaplan-Meier curve, log rank test, was generated to predict the overall survival of mice with RH30-SR Vector and RH30-SR MyoD Δ tumors, n = 5. (F) RH30-SR Vector and RH30-SR MyoD Δ cells were co-cultured with activated macrophages (0:1, 10:1, and 20:1) and cell survival was subsequently scored by trypan blue exclusion and compared to baseline cell death, analyzed with a two-way ANOVA with Sidak’s multiple comparisons with an interaction of p = 0.0133, n = 3. (G) RH30-SR Vector and RH30-SR MyoD Δ cells were treated with 0 or 10 ng/mL of TNF and 0, 10, or 50 nM of sodium nitroprusside (SNP) and cell survival was subsequently scored and analyzed as in (F), with an interaction of p = 0.0003, n = 3. (H) RH30-SR cells were administered to SCID mice and tumors that formed were subsequently injected with lentiviruses expressing OKMS factors or GFP control. Mice were then treated with 2 doses of vincristine (1.0 mg/kg; days 0 and 7; arrowheads) and tumor volumes were continually measured, n = 5. Data with error bars are depicted as mean ± SEM, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. See also .

Techniques: In Vivo, Infection, Plasmid Preparation, Control, Expressing, Clone Assay, Injection, Generated, Cell Culture

MyoD suppresses death genes through DNA methyltransferases (A) Volcano plot from transcriptomic analysis representing downregulated (blue, n = 1358) and upregulated genes (red, n = 1116) from RH30-SR MyoD Δ cells compared to RH30-SR cells (fold change >25%, FDR <0.05). (B) GO analysis of upregulated genes in RH30-SR MyoD Δ cells compared to RH30-SR cells. (C) Volcano plot from ATAC-seq analysis representing differentially accessible chromatin—closed (blue) and open (red)—in RH30-SR MyoD Δ cells compared to RH30-SR cells (fold change >25%, FDR <0.05). (D) Cell death was measured following the treatment of RH30-SR cells with decitabine (1 μM) and TNF (5 ng/mL) compared to RH30-SR control cells treated with DMSO and PBS. Significance was determined through two-way ANOVA analysis, with Sidak’s multiple comparisons, n = 3. (E) Expression levels of DNMT1, DNMT3A, and DNMT3B in RH30 MyoD Δ and RH30 Myf5 Δ compared to RH30-Vector control cells analyzed by one-way ANOVA with Dunnett’s multiple comparisons, n = 3. (F) Western blots of MyoD, DNMT1, DNMT3A, and DNMT3B in RH30-SR Vector or MyoD Δ cells, using α−tubulin as a loading control. Arrowheads point to respective DNMTs. (G) MyoD ChIP-seq data identifying enrichment peaks on the DNMT3A gene from FN (RD) and FP (RH4) RMS cells, graphed above representative ATAC-seq data revealing loss of chromatin accessibility signal in highlighted regions of interest in RH30 MyoD Δ (purple) compared to RH30 Vector cells (blue) with accompanying composite plot of ATAC-seq signals of both conditions, n = 3. Signal is represented as reads per million mapped reads (RPM). (H) Visualization of MyoD ChIP-seq data with enrichment peaks identified as S1-S3 on DNMT1, DNMT3A and DNMT3B genes in FN (RD) and FP (RH4) RMS cells. (I) MyoD ChIP analysis performed, as percent of input, on regions S1–S3 in DNMT1, DNMT3A and DNMT3B genes, n = 2. Data with error bars are depicted as mean ± SEM, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. See also .

Journal: iScience

Article Title: MyoD is essential in rhabdomyosarcoma by promoting survival through differentiation and CYLD

doi: 10.1016/j.isci.2025.113149

Figure Lengend Snippet: MyoD suppresses death genes through DNA methyltransferases (A) Volcano plot from transcriptomic analysis representing downregulated (blue, n = 1358) and upregulated genes (red, n = 1116) from RH30-SR MyoD Δ cells compared to RH30-SR cells (fold change >25%, FDR <0.05). (B) GO analysis of upregulated genes in RH30-SR MyoD Δ cells compared to RH30-SR cells. (C) Volcano plot from ATAC-seq analysis representing differentially accessible chromatin—closed (blue) and open (red)—in RH30-SR MyoD Δ cells compared to RH30-SR cells (fold change >25%, FDR <0.05). (D) Cell death was measured following the treatment of RH30-SR cells with decitabine (1 μM) and TNF (5 ng/mL) compared to RH30-SR control cells treated with DMSO and PBS. Significance was determined through two-way ANOVA analysis, with Sidak’s multiple comparisons, n = 3. (E) Expression levels of DNMT1, DNMT3A, and DNMT3B in RH30 MyoD Δ and RH30 Myf5 Δ compared to RH30-Vector control cells analyzed by one-way ANOVA with Dunnett’s multiple comparisons, n = 3. (F) Western blots of MyoD, DNMT1, DNMT3A, and DNMT3B in RH30-SR Vector or MyoD Δ cells, using α−tubulin as a loading control. Arrowheads point to respective DNMTs. (G) MyoD ChIP-seq data identifying enrichment peaks on the DNMT3A gene from FN (RD) and FP (RH4) RMS cells, graphed above representative ATAC-seq data revealing loss of chromatin accessibility signal in highlighted regions of interest in RH30 MyoD Δ (purple) compared to RH30 Vector cells (blue) with accompanying composite plot of ATAC-seq signals of both conditions, n = 3. Signal is represented as reads per million mapped reads (RPM). (H) Visualization of MyoD ChIP-seq data with enrichment peaks identified as S1-S3 on DNMT1, DNMT3A and DNMT3B genes in FN (RD) and FP (RH4) RMS cells. (I) MyoD ChIP analysis performed, as percent of input, on regions S1–S3 in DNMT1, DNMT3A and DNMT3B genes, n = 2. Data with error bars are depicted as mean ± SEM, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. See also .

Techniques: Control, Expressing, Plasmid Preparation, Western Blot, ChIP-sequencing

Identification of CYLD as a target of MyoD mediating RMS survival (A) qPCR analysis of 77 death related genes in RH30-SR MyoD Δ cells compared to Vector control, represented as technical duplicates. Arrowhead denotes the top 25 expressed genes, n = 1. (B) Illustration depicting a targeted CRISPR/Cas9 screen to identify genes involved in mediating survival of RH30-SR MyoD Δ RMS cells, created with BioRender.com . (C) PCR results from enrichment of targeted sgRNA guides in RH30-SR MyoD Δ cells infected with 0.5 virus particles per cell following four cycles of TNF treatment for 24 h compared to control untreated cells, represented as technical duplicates, n = 1. (D) RNA and protein levels of CYLD from myoblasts and myotubes compared to FN/ERMS and FP/ARMS tumors, from the St. Jude Pediatric Tumor Gene Expression Dataset. Dotted line denotes the maximum expression of CYLD from cultured muscle cells. (E and F) CYLD expression in RH30 (E) and RH5 (F) cells following treatment of decitabine compared to DMSO control, n = 3. Data with error bars are depicted as mean ± SEM, ∗ p < 0.05; ∗∗ p < 0.01. See also .

Journal: iScience

Article Title: MyoD is essential in rhabdomyosarcoma by promoting survival through differentiation and CYLD

doi: 10.1016/j.isci.2025.113149

Figure Lengend Snippet: Identification of CYLD as a target of MyoD mediating RMS survival (A) qPCR analysis of 77 death related genes in RH30-SR MyoD Δ cells compared to Vector control, represented as technical duplicates. Arrowhead denotes the top 25 expressed genes, n = 1. (B) Illustration depicting a targeted CRISPR/Cas9 screen to identify genes involved in mediating survival of RH30-SR MyoD Δ RMS cells, created with BioRender.com . (C) PCR results from enrichment of targeted sgRNA guides in RH30-SR MyoD Δ cells infected with 0.5 virus particles per cell following four cycles of TNF treatment for 24 h compared to control untreated cells, represented as technical duplicates, n = 1. (D) RNA and protein levels of CYLD from myoblasts and myotubes compared to FN/ERMS and FP/ARMS tumors, from the St. Jude Pediatric Tumor Gene Expression Dataset. Dotted line denotes the maximum expression of CYLD from cultured muscle cells. (E and F) CYLD expression in RH30 (E) and RH5 (F) cells following treatment of decitabine compared to DMSO control, n = 3. Data with error bars are depicted as mean ± SEM, ∗ p < 0.05; ∗∗ p < 0.01. See also .

Techniques: Plasmid Preparation, Control, CRISPR, Infection, Virus, Gene Expression, Expressing, Cell Culture

CYLD mediates TNF-induced cell death in RMS cells lacking MyoD (A) CYLD was depleted in RH30-SR MyoD Δ cells using CRISPR/Cas9 gene editing, and a western blot was performed to probe for CYLD expression. (B) RH30-SR MyoD Δ Vector and RH30-SR MyoD Δ CYLD Δ cells were treated with PBS or TNF for 24 h and cell viability was observed by phase contrast microscopy, scale bar denotes 100 μm. (C) Similar conditions as in (B), but cells were analyzed for cell death by Annexin V and flow cytometry, n = 3. (D) RH30-SR CYLD Δ cells were treated with decitabine and TNF, n = 2, and cell death was compared to Vector control cells, n = 3. (E) RH30-SR MyoD Δ cells were treated with TNF at indicated times and probed by Western for pRIPK1 and total RIPK1, with α−tubulin used as a loading control. (F) RH30-SR MyoD Δ Vector and RH30-SR MyoD Δ CYLD Δ cells were treated with100 nM of RIPK1 inhibitor GSK963 (RIPKi) or DMSO in the presence of TNF for 24 h and analyzed for cell death. Significance was determined through two-way ANOVA analysis, with Sidak’s multiple comparisons. The interaction between groups was not significant, n = 3. (G) NF-κB transcriptional activity was measured in RH30-SR Vector, RH30-SR MyoD Δ , RH30-SR MyoD Δ Vector, and RH30-SR MyoD Δ CYLD Δ cells using an NF-κB-Luciferase reporter plasmid in the presence of TNF compared to PBS as a control. C2C12 and RH30 MyoD Δ were used as control cell lines, n = 3. (H) qPCR assays were performed probing for NF-κB regulated genes A20, cIAP2, and TNF in RH30, RH30-SR MyoD, and RH30-SR MyoD CYLD Δ cells treated with TNF or PBS used as a control, n = 3. Data with error bars are depicted as mean ± SEM, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. See also .

Journal: iScience

Article Title: MyoD is essential in rhabdomyosarcoma by promoting survival through differentiation and CYLD

doi: 10.1016/j.isci.2025.113149

Figure Lengend Snippet: CYLD mediates TNF-induced cell death in RMS cells lacking MyoD (A) CYLD was depleted in RH30-SR MyoD Δ cells using CRISPR/Cas9 gene editing, and a western blot was performed to probe for CYLD expression. (B) RH30-SR MyoD Δ Vector and RH30-SR MyoD Δ CYLD Δ cells were treated with PBS or TNF for 24 h and cell viability was observed by phase contrast microscopy, scale bar denotes 100 μm. (C) Similar conditions as in (B), but cells were analyzed for cell death by Annexin V and flow cytometry, n = 3. (D) RH30-SR CYLD Δ cells were treated with decitabine and TNF, n = 2, and cell death was compared to Vector control cells, n = 3. (E) RH30-SR MyoD Δ cells were treated with TNF at indicated times and probed by Western for pRIPK1 and total RIPK1, with α−tubulin used as a loading control. (F) RH30-SR MyoD Δ Vector and RH30-SR MyoD Δ CYLD Δ cells were treated with100 nM of RIPK1 inhibitor GSK963 (RIPKi) or DMSO in the presence of TNF for 24 h and analyzed for cell death. Significance was determined through two-way ANOVA analysis, with Sidak’s multiple comparisons. The interaction between groups was not significant, n = 3. (G) NF-κB transcriptional activity was measured in RH30-SR Vector, RH30-SR MyoD Δ , RH30-SR MyoD Δ Vector, and RH30-SR MyoD Δ CYLD Δ cells using an NF-κB-Luciferase reporter plasmid in the presence of TNF compared to PBS as a control. C2C12 and RH30 MyoD Δ were used as control cell lines, n = 3. (H) qPCR assays were performed probing for NF-κB regulated genes A20, cIAP2, and TNF in RH30, RH30-SR MyoD, and RH30-SR MyoD CYLD Δ cells treated with TNF or PBS used as a control, n = 3. Data with error bars are depicted as mean ± SEM, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. See also .

Techniques: CRISPR, Western Blot, Expressing, Plasmid Preparation, Microscopy, Flow Cytometry, Control, Activity Assay, Luciferase

Effects of SLU-PP-332 treatment in myoblasts from inactive subjects on cell viability, cytotoxicity, oxidative stress, and senescence β-galactosidase activity (SA- β-gal). (a–d) : Immunofluorescence for Pax7 and MyoD in myoblasts: (a) Nuclei are stained with DAPI (blue); (b) Immunostaining for Pax7 (red); (c) Immunostaining for MyoD (green); (d) Merge for Pax7 and MyoD signals. 40× images, scale bar represents 100 μm. (e) MTS assay: the half inhibitory concentration (IC50) was obtained at a dosage between 1 × 10 −3 M and 2.5 × 10 −3 M (n = 9 from N = 3 experiments). (f) Lactate dehydrogenase (LDH) cytotoxicity assay: significant reduction of 16.1% of cell damage in SLU-PP-332-treated myoblasts (Inactive_T) compared with untreated cells (Inactive_NT) (p < 0.0001) (n = 25 from N = 5 experiments). (g) Intracellular reactive oxygen species (ROS) levels: significant reduction of 37.7% of oxidative stress in SLU-PP-332-treated myoblasts (Inactive_T) compared with untreated cells (Inactive_NT) (p < 0.0001) (n = 25 from N = 5 experiments). (h) Reduced glutathione (GSH) assay: significant increase of 117.4% in intracellular GSH levels in SLU-PP-332-treated myoblasts (Inactive_T) compared with untreated cells (Inactive_NT) (p < 0.0001) (n = 25 from N = 5 experiments). (i) SA-β-gal assay: significant reduction of 26.1% of enzymatic activity in SLU-PP-332-treated myoblasts (Inactive_T) compared with untreated cells (Inactive_NT) (p < 0.0001) (n = 25 from N = 5 experiments).

Journal: Frontiers in Physiology

Article Title: Targeting ERRs to counteract age-related muscle atrophy associated with physical inactivity: a pilot study

doi: 10.3389/fphys.2025.1616693

Figure Lengend Snippet: Effects of SLU-PP-332 treatment in myoblasts from inactive subjects on cell viability, cytotoxicity, oxidative stress, and senescence β-galactosidase activity (SA- β-gal). (a–d) : Immunofluorescence for Pax7 and MyoD in myoblasts: (a) Nuclei are stained with DAPI (blue); (b) Immunostaining for Pax7 (red); (c) Immunostaining for MyoD (green); (d) Merge for Pax7 and MyoD signals. 40× images, scale bar represents 100 μm. (e) MTS assay: the half inhibitory concentration (IC50) was obtained at a dosage between 1 × 10 −3 M and 2.5 × 10 −3 M (n = 9 from N = 3 experiments). (f) Lactate dehydrogenase (LDH) cytotoxicity assay: significant reduction of 16.1% of cell damage in SLU-PP-332-treated myoblasts (Inactive_T) compared with untreated cells (Inactive_NT) (p < 0.0001) (n = 25 from N = 5 experiments). (g) Intracellular reactive oxygen species (ROS) levels: significant reduction of 37.7% of oxidative stress in SLU-PP-332-treated myoblasts (Inactive_T) compared with untreated cells (Inactive_NT) (p < 0.0001) (n = 25 from N = 5 experiments). (h) Reduced glutathione (GSH) assay: significant increase of 117.4% in intracellular GSH levels in SLU-PP-332-treated myoblasts (Inactive_T) compared with untreated cells (Inactive_NT) (p < 0.0001) (n = 25 from N = 5 experiments). (i) SA-β-gal assay: significant reduction of 26.1% of enzymatic activity in SLU-PP-332-treated myoblasts (Inactive_T) compared with untreated cells (Inactive_NT) (p < 0.0001) (n = 25 from N = 5 experiments).

Article Snippet: Briefly, after fixation in 4% paraformaldehyde dissolved in 0.9% saline solution for 30 min, cell cultures were pretreated with EDTA citrate, pH 7.8 for 20 min at 95 C, and incubated for 1 h with rabbit polyclonal anti-Pax7 antibody (dilution 1:100; ab187339, AbCam, Cambridge, United Kingdom), mouse monoclonal anti-MyoD antibody (dilution 1:100; InvitrogenTM, ThermoFisher Scientific, United States), rabbit polyclonal anti-ERRα antibody (dilution 1:100; A90033, antibodies.com, Stockholm, Sweden), or mouse monoclonal anti-MyHC antibody (diluition 1:100; ab51263, AbCam, Cambridge, United Kingdom).

Techniques: Activity Assay, Immunofluorescence, Staining, Immunostaining, MTS Assay, Concentration Assay, LDH Cytotoxicity Assay, GSH Assay

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