Structured Review

Thermo Fisher myf5
Differentiation of myoblasts into myotubes is inhibited by SVF cells and HGF. a Immunofluorescence staining of fast type myosin heavy chain (green) in C2C12 mouse myoblasts after 4 days in growth medium (GM), differentiation medium (DM), indirect co-culture with SVF cells in DM (SVF), and DM supplemented with 10 ng/ml HGF (HGF). Cells were counterstained with DAPI (blue). Bar = 200 μm. The expression of myogenic regulatory factor 5 ( <t>Myf5</t> ), slow type myosin heavy chain ( Myhc1 ), and fast type myosin heavy chain ( Myhc2 ) mRNA in C2C12 myoblasts after b 7 days or c 14 days of differentiation in the various culture conditions. Western blot of fast type myosin heavy chain (MyHC2) protein is also shown. β-actin was used as a loading control. Statistical analysis was performed using one-way ANOVA with post hoc test (Bonferroni correction). Results are presented as mean ± standard deviation. n = 3. * P
Myf5, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "The adipose tissue stromal vascular fraction secretome enhances the proliferation but inhibits the differentiation of myoblasts"

Article Title: The adipose tissue stromal vascular fraction secretome enhances the proliferation but inhibits the differentiation of myoblasts

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-018-1096-6

Differentiation of myoblasts into myotubes is inhibited by SVF cells and HGF. a Immunofluorescence staining of fast type myosin heavy chain (green) in C2C12 mouse myoblasts after 4 days in growth medium (GM), differentiation medium (DM), indirect co-culture with SVF cells in DM (SVF), and DM supplemented with 10 ng/ml HGF (HGF). Cells were counterstained with DAPI (blue). Bar = 200 μm. The expression of myogenic regulatory factor 5 ( Myf5 ), slow type myosin heavy chain ( Myhc1 ), and fast type myosin heavy chain ( Myhc2 ) mRNA in C2C12 myoblasts after b 7 days or c 14 days of differentiation in the various culture conditions. Western blot of fast type myosin heavy chain (MyHC2) protein is also shown. β-actin was used as a loading control. Statistical analysis was performed using one-way ANOVA with post hoc test (Bonferroni correction). Results are presented as mean ± standard deviation. n = 3. * P
Figure Legend Snippet: Differentiation of myoblasts into myotubes is inhibited by SVF cells and HGF. a Immunofluorescence staining of fast type myosin heavy chain (green) in C2C12 mouse myoblasts after 4 days in growth medium (GM), differentiation medium (DM), indirect co-culture with SVF cells in DM (SVF), and DM supplemented with 10 ng/ml HGF (HGF). Cells were counterstained with DAPI (blue). Bar = 200 μm. The expression of myogenic regulatory factor 5 ( Myf5 ), slow type myosin heavy chain ( Myhc1 ), and fast type myosin heavy chain ( Myhc2 ) mRNA in C2C12 myoblasts after b 7 days or c 14 days of differentiation in the various culture conditions. Western blot of fast type myosin heavy chain (MyHC2) protein is also shown. β-actin was used as a loading control. Statistical analysis was performed using one-way ANOVA with post hoc test (Bonferroni correction). Results are presented as mean ± standard deviation. n = 3. * P

Techniques Used: Immunofluorescence, Staining, Co-Culture Assay, Expressing, Western Blot, Standard Deviation

2) Product Images from "The adipose tissue stromal vascular fraction secretome enhances the proliferation but inhibits the differentiation of myoblasts"

Article Title: The adipose tissue stromal vascular fraction secretome enhances the proliferation but inhibits the differentiation of myoblasts

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-018-1096-6

Differentiation of myoblasts into myotubes is inhibited by SVF cells and HGF. a Immunofluorescence staining of fast type myosin heavy chain (green) in C2C12 mouse myoblasts after 4 days in growth medium (GM), differentiation medium (DM), indirect co-culture with SVF cells in DM (SVF), and DM supplemented with 10 ng/ml HGF (HGF). Cells were counterstained with DAPI (blue). Bar = 200 μm. The expression of myogenic regulatory factor 5 ( Myf5 ), slow type myosin heavy chain ( Myhc1 ), and fast type myosin heavy chain ( Myhc2 ) mRNA in C2C12 myoblasts after b 7 days or c 14 days of differentiation in the various culture conditions. Western blot of fast type myosin heavy chain (MyHC2) protein is also shown. β-actin was used as a loading control. Statistical analysis was performed using one-way ANOVA with post hoc test (Bonferroni correction). Results are presented as mean ± standard deviation. n = 3. * P
Figure Legend Snippet: Differentiation of myoblasts into myotubes is inhibited by SVF cells and HGF. a Immunofluorescence staining of fast type myosin heavy chain (green) in C2C12 mouse myoblasts after 4 days in growth medium (GM), differentiation medium (DM), indirect co-culture with SVF cells in DM (SVF), and DM supplemented with 10 ng/ml HGF (HGF). Cells were counterstained with DAPI (blue). Bar = 200 μm. The expression of myogenic regulatory factor 5 ( Myf5 ), slow type myosin heavy chain ( Myhc1 ), and fast type myosin heavy chain ( Myhc2 ) mRNA in C2C12 myoblasts after b 7 days or c 14 days of differentiation in the various culture conditions. Western blot of fast type myosin heavy chain (MyHC2) protein is also shown. β-actin was used as a loading control. Statistical analysis was performed using one-way ANOVA with post hoc test (Bonferroni correction). Results are presented as mean ± standard deviation. n = 3. * P

Techniques Used: Immunofluorescence, Staining, Co-Culture Assay, Expressing, Western Blot, Standard Deviation

3) Product Images from "Low-Intensity Extracorporeal Shock Wave Therapy Promotes Myogenesis Through PERK/ATF4 Pathway"

Article Title: Low-Intensity Extracorporeal Shock Wave Therapy Promotes Myogenesis Through PERK/ATF4 Pathway

Journal: Neurourology and urodynamics

doi: 10.1002/nau.23380

Characterization of uMDSCs and myotube formation (a) Image of uMDSCs from P0 to P1 under microscope. (b) Flow cytometry analysis of uMDSCs surface markers revealed expression of CD34, Int-7α, CD56, Myf5, pax7, CD105, Int-7α/CD56, CD34/CD56, Myf5/pax7, and myf5/CD105. (c) Two groups of uMDSCs were immunostained using MHC for myotube formation. (cont=control, HS=2% horse serum)
Figure Legend Snippet: Characterization of uMDSCs and myotube formation (a) Image of uMDSCs from P0 to P1 under microscope. (b) Flow cytometry analysis of uMDSCs surface markers revealed expression of CD34, Int-7α, CD56, Myf5, pax7, CD105, Int-7α/CD56, CD34/CD56, Myf5/pax7, and myf5/CD105. (c) Two groups of uMDSCs were immunostained using MHC for myotube formation. (cont=control, HS=2% horse serum)

Techniques Used: Microscopy, Flow Cytometry, Cytometry, Expressing

Li-ESWT increased the expression of MHC and Myf5 in L6 cells, and this response was attenuated by GSK2656157 (a) Expression of MHC and Myf5 with usage of Li-ESWT and GSK2656157 (n=3 in triplicates). (b) The expression level of MHC increased significantly with Li-ESWT and could be blocked by GSK2656157 (*p
Figure Legend Snippet: Li-ESWT increased the expression of MHC and Myf5 in L6 cells, and this response was attenuated by GSK2656157 (a) Expression of MHC and Myf5 with usage of Li-ESWT and GSK2656157 (n=3 in triplicates). (b) The expression level of MHC increased significantly with Li-ESWT and could be blocked by GSK2656157 (*p

Techniques Used: Expressing

4) Product Images from "A Gingiva-Derived Mesenchymal Stem Cell-Laden Porcine Small Intestinal Submucosa Extracellular Matrix Construct Promotes Myomucosal Regeneration of the Tongue"

Article Title: A Gingiva-Derived Mesenchymal Stem Cell-Laden Porcine Small Intestinal Submucosa Extracellular Matrix Construct Promotes Myomucosal Regeneration of the Tongue

Journal: Tissue Engineering. Part A

doi: 10.1089/ten.tea.2016.0342

Engraftment of Transplanted GMSCs at the Wound Site and Expression of Myogenic Transcriptional Factors. (A) H E staining results of cross section of specimen taken after GMSC/SIS-ECM construction implantation at 2 weeks. Blue circle indicates the area of myomucosal regeneration. Muscle regeneration was not identified until 2 weeks in all groups (original magnification ×4). The distribution of PKH26 prelabeled GMSCs at the circled regeneration area was observed under a fluorescence microscope (original magnification ×10). The nuclei were counterstained with DAPI. Coexpression of MyoD ( green ) (B) , Myf5 ( green ) (C) , PAX7 ( green ) (D) , and PKH26 ( red ) in GMSCs at local defect area after 2 weeks transplantation was determined by immunostaining and observed under a fluorescence microscope. The nuclei were counterstained with DAPI (original magnification ×20).
Figure Legend Snippet: Engraftment of Transplanted GMSCs at the Wound Site and Expression of Myogenic Transcriptional Factors. (A) H E staining results of cross section of specimen taken after GMSC/SIS-ECM construction implantation at 2 weeks. Blue circle indicates the area of myomucosal regeneration. Muscle regeneration was not identified until 2 weeks in all groups (original magnification ×4). The distribution of PKH26 prelabeled GMSCs at the circled regeneration area was observed under a fluorescence microscope (original magnification ×10). The nuclei were counterstained with DAPI. Coexpression of MyoD ( green ) (B) , Myf5 ( green ) (C) , PAX7 ( green ) (D) , and PKH26 ( red ) in GMSCs at local defect area after 2 weeks transplantation was determined by immunostaining and observed under a fluorescence microscope. The nuclei were counterstained with DAPI (original magnification ×20).

Techniques Used: Expressing, Staining, Fluorescence, Microscopy, Transplantation Assay, Immunostaining

Increased expression of Myf5 in wounded tongue areas with transplantation of GMSC/SIS-ECM in comparison with SIS-ECM alone and defect control. (A) Expression of Myf5 ( green ) at local defect area 2 weeks after surgery in control, SIS-ECM only, and GMSC/SIS-ECM construct groups was determined by immunostaining and observed under a fluorescence microscope. The nuclei were counterstained with DAPI (original magnification ×10; ×20). (B) The integrated immunofluorescence density of a ROI was quantified using Olympus cellSens imaging software. (C) The expression of Myf5 protein at the injured areas of the tongue was determined by Western blot analysis.
Figure Legend Snippet: Increased expression of Myf5 in wounded tongue areas with transplantation of GMSC/SIS-ECM in comparison with SIS-ECM alone and defect control. (A) Expression of Myf5 ( green ) at local defect area 2 weeks after surgery in control, SIS-ECM only, and GMSC/SIS-ECM construct groups was determined by immunostaining and observed under a fluorescence microscope. The nuclei were counterstained with DAPI (original magnification ×10; ×20). (B) The integrated immunofluorescence density of a ROI was quantified using Olympus cellSens imaging software. (C) The expression of Myf5 protein at the injured areas of the tongue was determined by Western blot analysis.

Techniques Used: Expressing, Transplantation Assay, Construct, Immunostaining, Fluorescence, Microscopy, Immunofluorescence, Imaging, Software, Western Blot

Related Articles

Microarray:

Article Title: A CreER based random induction strategy for modeling translocation-associated sarcomas in mice
Article Snippet: .. We have previously generated microarray data from mouse synovial sarcoma-like tumors in Myf5-Cre+/− /SSM2+/− mice and control wild type skeletal muscles using the same Affymetrix platform. .. We next searched the Gene Expression Omnibus (GEO) database for availability of microarray data generated on Affymetrix mouse 430 2.0 genechip (same platform to reduce cross-platform issues) from other mouse models of tumors and found the following datasets matching our criteria; Kaposi's Sarcoma , thymic lymphomas , mammary tumors from two distinct models ( ) , multiple myeloma , and osteosarcoma ( ).

Generated:

Article Title: A CreER based random induction strategy for modeling translocation-associated sarcomas in mice
Article Snippet: .. We have previously generated microarray data from mouse synovial sarcoma-like tumors in Myf5-Cre+/− /SSM2+/− mice and control wild type skeletal muscles using the same Affymetrix platform. .. We next searched the Gene Expression Omnibus (GEO) database for availability of microarray data generated on Affymetrix mouse 430 2.0 genechip (same platform to reduce cross-platform issues) from other mouse models of tumors and found the following datasets matching our criteria; Kaposi's Sarcoma , thymic lymphomas , mammary tumors from two distinct models ( ) , multiple myeloma , and osteosarcoma ( ).

Mouse Assay:

Article Title: A CreER based random induction strategy for modeling translocation-associated sarcomas in mice
Article Snippet: .. We have previously generated microarray data from mouse synovial sarcoma-like tumors in Myf5-Cre+/− /SSM2+/− mice and control wild type skeletal muscles using the same Affymetrix platform. .. We next searched the Gene Expression Omnibus (GEO) database for availability of microarray data generated on Affymetrix mouse 430 2.0 genechip (same platform to reduce cross-platform issues) from other mouse models of tumors and found the following datasets matching our criteria; Kaposi's Sarcoma , thymic lymphomas , mammary tumors from two distinct models ( ) , multiple myeloma , and osteosarcoma ( ).

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    Thermo Fisher gene exp myf5 hs00271574 m1
    SNAIL is a direct transcriptional repressor of <t>MYF5</t> expression and regulator of MYOD activity. a SNAIL silencing in RH30 cells induces MYOD binding to GC rich E-Box sequences. Nuclear protein extracts from both undifferentiated RH30 cells and RH30 cells differentiated for 7 days in medium supplemented with 2% HS were used in TransAM MyoD DNA-binding ELISA. To monitor the specificity of the assay, the wild-type consensus oligonucleotide (wt) and mutated (mut) sequences were used as competitors for MYOD binding from cell extracts; n = 3. b SNAIL binds to the MYF5 and E-cadherin promoters in RH30 cells. The promoter of MYF5 (~1000 bb) was screened for putative SNAIL transcription factor binding sites and the results were validated by Chip Assay. The images depict one representative result of the ChIP assay. Proteins bound to DNA were immunoprecipitated with the anti-SNAIL antibody, negative IgG control, positive histone H3 control and input DNA control was analyzed. Fragments of the MYF5 and E-cadherin promoters were amplified by PCR and visualized on agarose gels stained with ethidium bromide. c SNAIL silencing by siRNA in RH30 WT cells induces MYF5 promoter activation and luciferase expression when cells are transfected with MYF5@pNL plasmid (luciferase under control with MYF5 promoter), whereas MYOD silencing by siRNA does not exert any effect. SNAIL and MYOD compete for binding to MYF5 promoter in RH30 shSNAIL cells ( d ), RH41 WT ( e ), and RH41 shSNAIL cells ( f ), when cells are transfected with MYF5@pNL plasmid and siRNA against SNAIL, MYOD or siSCR (scrambled siRNA). The data were normalized to mCherry fluorescence level in each well. pNL (luciferase plasmid without promoter) served as a negative control and Ubc@pNL plasmid (luciferase under control of ubiquitin C promoter) was a positive control. The data represent the mean ± SEM or are representative images of at least 3 independent experiments. * p
    Gene Exp Myf5 Hs00271574 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gene exp myf5 hs00271574 m1/product/Thermo Fisher
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    Thermo Fisher gene exp myf5 mm00435125 m1
    Tbx6 Induced and Maintained a Mesoderm Program in Mouse Fibroblasts (A and B) qRT-PCR for Mesp1 expression after transduction of individual (A) or combinatorial (B) factors after 1 week (n = 3, independent experiments). Data were normalized to the values in MEFs. (C) Immunocytochemistry for Mesp1Cre-GFP and DAPI with quantification of the numbers of Mesp1Cre-GFP + colonies (n = 3, independent experiments). Tbx6 induced Mesp1Cre-GFP expression after 2 weeks of transduction. (D) Immunocytochemistry and quantification of the numbers of T + colonies (n = 3, independent experiments). Immunocytochemistry demonstrated that T protein was expressed in Tbx6transduced mouse embryonic fibroblast nuclei after 4 weeks. (F) FACS analyses showed that Tbx6transduced cells expressed cell-surface markers Flk1 and PDGFRα (n = 3, independent experiments). Time course of mRNA expression in MEFs transduced with Tbx6, as determined by qRT-PCR. Gene expression for mesoderm ( Mesp1 , T , Flk1 ), CPCs ( Isl1 , Nkx2.5 , Gata4 ), CMs ( Tnnt2 , Myh6 ), SMCs ( Myh11 ), ECs ( Pecam1 ), and skeletal muscle ( Pax3 , Pax7 , Myod1 , <t>Myf5</t> ) genes were determined. Tbx6-transduced cells expressed mesoderm genes, but not those associated with cardiovascular differentiation and skeletal muscle genes. Data were normalized to the values on day 0. All data are presented as mean ± SD. **p
    Gene Exp Myf5 Mm00435125 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gene exp myf5 hs00929416 g1
    BETi have a memory effect on DUX4 that is mediated by HDACs. a – c BETi cause sustained DUX4 repression. a Experimental timeline. Subconfluent MB200 FSHD2 myoblasts were treated with 1 μM of the BETi I-BET762 (I-BET) on day 0 (D0) for 8, 24, 48, or 72 h and gene expression analyzed on day 3 (D3). b DUX4 target gene mRNA levels after treatment as in a . c Expression of the myoblast lineage genes MYOD1 and <t>MYF5</t> and the epigenetic modifier SMCHD1 after treatment as in a . d – e HDACi block the BETi-mediated memory effect. d Levels of the DUX4 target gene ZSCAN4 in MB200 FSHD2 myoblasts that were treated with the indicated compounds (DMSO control, 1 μM I-BET762, 2.5 μM MS-275, 2.5 μM MGCD0103) for 24 h and then cultured in fresh media for an additional 48 h before harvest. e ZSCAN4 expression after 72 h of continuous exposure to the indicated compounds (DMSO control, 1 μM I-BET762, 2 μM RGFP109) in MB200 FSHD2 myoblasts. Error bars indicate the standard deviation from the mean of three biological replicates. p values were calculated using a one-way analysis of variance with Dunnett’s post test. * p
    Gene Exp Myf5 Hs00929416 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SNAIL is a direct transcriptional repressor of MYF5 expression and regulator of MYOD activity. a SNAIL silencing in RH30 cells induces MYOD binding to GC rich E-Box sequences. Nuclear protein extracts from both undifferentiated RH30 cells and RH30 cells differentiated for 7 days in medium supplemented with 2% HS were used in TransAM MyoD DNA-binding ELISA. To monitor the specificity of the assay, the wild-type consensus oligonucleotide (wt) and mutated (mut) sequences were used as competitors for MYOD binding from cell extracts; n = 3. b SNAIL binds to the MYF5 and E-cadherin promoters in RH30 cells. The promoter of MYF5 (~1000 bb) was screened for putative SNAIL transcription factor binding sites and the results were validated by Chip Assay. The images depict one representative result of the ChIP assay. Proteins bound to DNA were immunoprecipitated with the anti-SNAIL antibody, negative IgG control, positive histone H3 control and input DNA control was analyzed. Fragments of the MYF5 and E-cadherin promoters were amplified by PCR and visualized on agarose gels stained with ethidium bromide. c SNAIL silencing by siRNA in RH30 WT cells induces MYF5 promoter activation and luciferase expression when cells are transfected with MYF5@pNL plasmid (luciferase under control with MYF5 promoter), whereas MYOD silencing by siRNA does not exert any effect. SNAIL and MYOD compete for binding to MYF5 promoter in RH30 shSNAIL cells ( d ), RH41 WT ( e ), and RH41 shSNAIL cells ( f ), when cells are transfected with MYF5@pNL plasmid and siRNA against SNAIL, MYOD or siSCR (scrambled siRNA). The data were normalized to mCherry fluorescence level in each well. pNL (luciferase plasmid without promoter) served as a negative control and Ubc@pNL plasmid (luciferase under control of ubiquitin C promoter) was a positive control. The data represent the mean ± SEM or are representative images of at least 3 independent experiments. * p

    Journal: Cell Death & Disease

    Article Title: SNAIL is a key regulator of alveolar rhabdomyosarcoma tumor growth and differentiation through repression of MYF5 and MYOD function

    doi: 10.1038/s41419-018-0693-8

    Figure Lengend Snippet: SNAIL is a direct transcriptional repressor of MYF5 expression and regulator of MYOD activity. a SNAIL silencing in RH30 cells induces MYOD binding to GC rich E-Box sequences. Nuclear protein extracts from both undifferentiated RH30 cells and RH30 cells differentiated for 7 days in medium supplemented with 2% HS were used in TransAM MyoD DNA-binding ELISA. To monitor the specificity of the assay, the wild-type consensus oligonucleotide (wt) and mutated (mut) sequences were used as competitors for MYOD binding from cell extracts; n = 3. b SNAIL binds to the MYF5 and E-cadherin promoters in RH30 cells. The promoter of MYF5 (~1000 bb) was screened for putative SNAIL transcription factor binding sites and the results were validated by Chip Assay. The images depict one representative result of the ChIP assay. Proteins bound to DNA were immunoprecipitated with the anti-SNAIL antibody, negative IgG control, positive histone H3 control and input DNA control was analyzed. Fragments of the MYF5 and E-cadherin promoters were amplified by PCR and visualized on agarose gels stained with ethidium bromide. c SNAIL silencing by siRNA in RH30 WT cells induces MYF5 promoter activation and luciferase expression when cells are transfected with MYF5@pNL plasmid (luciferase under control with MYF5 promoter), whereas MYOD silencing by siRNA does not exert any effect. SNAIL and MYOD compete for binding to MYF5 promoter in RH30 shSNAIL cells ( d ), RH41 WT ( e ), and RH41 shSNAIL cells ( f ), when cells are transfected with MYF5@pNL plasmid and siRNA against SNAIL, MYOD or siSCR (scrambled siRNA). The data were normalized to mCherry fluorescence level in each well. pNL (luciferase plasmid without promoter) served as a negative control and Ubc@pNL plasmid (luciferase under control of ubiquitin C promoter) was a positive control. The data represent the mean ± SEM or are representative images of at least 3 independent experiments. * p

    Article Snippet: Quantitative real-time PCR Gene expression was determined by qRT-PCR analysis using ABI PRISM 7300 Sequence Detection System or Quant Studio 7 Flex System (both from Applied Biosystems, Foster City, CA, USA), Blank qPCR Master Mix (EURx) and the indicated Taq-Man probes (Applied Biosystems): human: GAPDH (Hs99999905_m1), MYF5 (Hs00271574_m1), MYOD (Hs00159528_m1), MRF4 (Hs01547104_g1), MEF2A (Hs01050409_m1), MYOSTATIN (Hs00976237_m1), MYOGENIN (Hs01032275_m1), MYH2 (Hs00430042_m1), SNAI1 (Hs00195591_m1), HDAC1 (Hs02621185_s1) and HDAC2 (Hs00231032_m1).

    Techniques: Expressing, Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Polymerase Chain Reaction, Staining, Activation Assay, Luciferase, Transfection, Plasmid Preparation, Fluorescence, Negative Control, Positive Control

    SNAIL silencing in human myoblasts increases expression of myogenic factors. a SNAIL expression in human myoblasts diminishes with subsequent passages (qPCR, n = 4). b Human myoblasts were transfected with siRNA against SNAIL (siSNAIL) and with a scrambled siRNA sequence (siRNA) or they were treated with transfection reagent (mock). 24 h after transfection they were treated with a differentiating medium containing 2% HS for the next 48 h. SNAIL silencing was validated by Western blot. SNAIL silencing increased the myogenin and MyHC protein levels (Western blot). c Expression of SNAIL, MYF5, MYOD, MRF4, myogenin and MyHC was determined by qPCR with ΔCT quantification method and GAPDH as a housekeeping gene control. SNAIL silencing and differentiation induces expression of the analyzed myogenic factors. d SNAIL silencing increases the number of the myotubes with high MyHC expression and induces their fusion. The images are representative merged images of immunofluorescent staining for MyHC (MyHC: red; nuclei: Hoechst, blue) of myotubes. The scale bar represents 100 μm. Fusion index was calculated by expressing the number of nuclei within MyHC-positive myotubes with ≥2 nuclei as a percentage of the total nuclei; n = 4. e Mechanism of action of SNAIL transcription factor on myogenic differentiation of ARMS. Data on graphs are represented as mean ± SEM. * p

    Journal: Cell Death & Disease

    Article Title: SNAIL is a key regulator of alveolar rhabdomyosarcoma tumor growth and differentiation through repression of MYF5 and MYOD function

    doi: 10.1038/s41419-018-0693-8

    Figure Lengend Snippet: SNAIL silencing in human myoblasts increases expression of myogenic factors. a SNAIL expression in human myoblasts diminishes with subsequent passages (qPCR, n = 4). b Human myoblasts were transfected with siRNA against SNAIL (siSNAIL) and with a scrambled siRNA sequence (siRNA) or they were treated with transfection reagent (mock). 24 h after transfection they were treated with a differentiating medium containing 2% HS for the next 48 h. SNAIL silencing was validated by Western blot. SNAIL silencing increased the myogenin and MyHC protein levels (Western blot). c Expression of SNAIL, MYF5, MYOD, MRF4, myogenin and MyHC was determined by qPCR with ΔCT quantification method and GAPDH as a housekeeping gene control. SNAIL silencing and differentiation induces expression of the analyzed myogenic factors. d SNAIL silencing increases the number of the myotubes with high MyHC expression and induces their fusion. The images are representative merged images of immunofluorescent staining for MyHC (MyHC: red; nuclei: Hoechst, blue) of myotubes. The scale bar represents 100 μm. Fusion index was calculated by expressing the number of nuclei within MyHC-positive myotubes with ≥2 nuclei as a percentage of the total nuclei; n = 4. e Mechanism of action of SNAIL transcription factor on myogenic differentiation of ARMS. Data on graphs are represented as mean ± SEM. * p

    Article Snippet: Quantitative real-time PCR Gene expression was determined by qRT-PCR analysis using ABI PRISM 7300 Sequence Detection System or Quant Studio 7 Flex System (both from Applied Biosystems, Foster City, CA, USA), Blank qPCR Master Mix (EURx) and the indicated Taq-Man probes (Applied Biosystems): human: GAPDH (Hs99999905_m1), MYF5 (Hs00271574_m1), MYOD (Hs00159528_m1), MRF4 (Hs01547104_g1), MEF2A (Hs01050409_m1), MYOSTATIN (Hs00976237_m1), MYOGENIN (Hs01032275_m1), MYH2 (Hs00430042_m1), SNAI1 (Hs00195591_m1), HDAC1 (Hs02621185_s1) and HDAC2 (Hs00231032_m1).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Sequencing, Western Blot, Staining

    SNAIL expression is associated with RMS subtype and myogenic differentiation. a Expression of early and late myogenic factors in 158 RMS samples was estimated previously by microarray and deposited in GEO database with accession number: GSE92689 (ref. 20 ). SNAIL level negatively correlates with MYF5 level in RMS (Pearson correlation). b SNAIL level positively correlates with MYOD level in RMS (Pearson correlation). R—Pearson correlation coefficient; p—significance value ( c ) Increased SNAIL levels in RMS stage 2,3 and 4 compared to 1 were demonstrated in a group of 158 RMS samples (microarray data, GEO database GSE92689). The data are presented as Whisker plots min to max. d Increased SNAIL level in ARMS compared to ERMS was demonstrated in a group of 158 RMS samples (microarray data, GEO database GSE92689). The data are presented as Whisker plots min to max. e SNAIL expression was quantified by qPCR using the ΔCt quantification method and GAPDH as a housekeeping gene control in ARMS and ERMS cell lines and human primary myoblasts. The SNAIL level is increased in RH30 and RH41 ARMS cell lines compared to RD and RH18 ERMS cell lines, and the level is comparable with the SNAIL level in myoblasts at the early passage (undiff.), whereas in the differentiated myotubes (diff.) SNAIL level is more comparable with ERMS; n = 3. f Differentiation of RH30 cells by culture in medium supplemented with 2% HS for 7 days downregulates SNAIL expression at the mRNA level (qPCR) and protein level (Western blotting); n = 4. The data represent the mean ± SEM. * p

    Journal: Cell Death & Disease

    Article Title: SNAIL is a key regulator of alveolar rhabdomyosarcoma tumor growth and differentiation through repression of MYF5 and MYOD function

    doi: 10.1038/s41419-018-0693-8

    Figure Lengend Snippet: SNAIL expression is associated with RMS subtype and myogenic differentiation. a Expression of early and late myogenic factors in 158 RMS samples was estimated previously by microarray and deposited in GEO database with accession number: GSE92689 (ref. 20 ). SNAIL level negatively correlates with MYF5 level in RMS (Pearson correlation). b SNAIL level positively correlates with MYOD level in RMS (Pearson correlation). R—Pearson correlation coefficient; p—significance value ( c ) Increased SNAIL levels in RMS stage 2,3 and 4 compared to 1 were demonstrated in a group of 158 RMS samples (microarray data, GEO database GSE92689). The data are presented as Whisker plots min to max. d Increased SNAIL level in ARMS compared to ERMS was demonstrated in a group of 158 RMS samples (microarray data, GEO database GSE92689). The data are presented as Whisker plots min to max. e SNAIL expression was quantified by qPCR using the ΔCt quantification method and GAPDH as a housekeeping gene control in ARMS and ERMS cell lines and human primary myoblasts. The SNAIL level is increased in RH30 and RH41 ARMS cell lines compared to RD and RH18 ERMS cell lines, and the level is comparable with the SNAIL level in myoblasts at the early passage (undiff.), whereas in the differentiated myotubes (diff.) SNAIL level is more comparable with ERMS; n = 3. f Differentiation of RH30 cells by culture in medium supplemented with 2% HS for 7 days downregulates SNAIL expression at the mRNA level (qPCR) and protein level (Western blotting); n = 4. The data represent the mean ± SEM. * p

    Article Snippet: Quantitative real-time PCR Gene expression was determined by qRT-PCR analysis using ABI PRISM 7300 Sequence Detection System or Quant Studio 7 Flex System (both from Applied Biosystems, Foster City, CA, USA), Blank qPCR Master Mix (EURx) and the indicated Taq-Man probes (Applied Biosystems): human: GAPDH (Hs99999905_m1), MYF5 (Hs00271574_m1), MYOD (Hs00159528_m1), MRF4 (Hs01547104_g1), MEF2A (Hs01050409_m1), MYOSTATIN (Hs00976237_m1), MYOGENIN (Hs01032275_m1), MYH2 (Hs00430042_m1), SNAI1 (Hs00195591_m1), HDAC1 (Hs02621185_s1) and HDAC2 (Hs00231032_m1).

    Techniques: Expressing, Microarray, Whisker Assay, Real-time Polymerase Chain Reaction, Western Blot

    SNAIL silencing induces MYF5 expression in ARMS cells. a SNAIL silencing does not significantly affect MYOD expression at the mRNA (qPCR, n = 3) or protein level (Western blot) in undifferentiated RH30 cells and RH30 cells differentiated for 7 days in medium with 2% HS. b MYF5 is expressed in the nuclei of RH30 shSNAIL cells. The MYF5 protein level was visualized by Western blot and by immunofluorescent staining (red color), and the nuclei were visualized with DAPI (blue). The results are presented as merged images. The white scale bar represents 10 μm. c SNAIL silencing induces the expression of MYF5 mRNA in both undifferentiated RH30 cells and RH30 cells differentiated for 7 days in medium with 2% HS (qPCR, n = 3) d SNAIL silencing slightly induces the expression of MYF5 mRNA in both undifferentiated RH41 cells and RH41 cells differentiated for 7 days in medium with 2% HS (qPCR, n = 3). e Transient SNAIL silencing with siRNA in RH30 and RH41 ARMS cell lines slightly induces MYF5 expression. The cells were transfected with siRNA against SNAIL (siSNAIL) and a scrambled siRNA sequence (siRNA). Twenty-four hours after transfection, the cells were treated with differentiating medium containing 2% HS for the following 48 h. MYF5 levels were validated by qPCR; n = 3. f Transfection with pre-miR-30a-5p in RH30 cells induces MYF5 expression by downregulation of SNAIL protein. RH30 cells were transfected with the miRNA precursor pre-miR-30a-5p and pre-miR negative control (miR-neg-ctrl). Twenty-four hours after transfection, the cells were treated with differentiating medium containing 2% HS for the following 48 h. miR-30a-5p expression relative to U6 snRNA and MYF5, MYOD expression relative to GAPDH were validated by qPCR; n = 4. SNAIL protein level was validated by Western blot. The data represent the mean ± SEM. * p

    Journal: Cell Death & Disease

    Article Title: SNAIL is a key regulator of alveolar rhabdomyosarcoma tumor growth and differentiation through repression of MYF5 and MYOD function

    doi: 10.1038/s41419-018-0693-8

    Figure Lengend Snippet: SNAIL silencing induces MYF5 expression in ARMS cells. a SNAIL silencing does not significantly affect MYOD expression at the mRNA (qPCR, n = 3) or protein level (Western blot) in undifferentiated RH30 cells and RH30 cells differentiated for 7 days in medium with 2% HS. b MYF5 is expressed in the nuclei of RH30 shSNAIL cells. The MYF5 protein level was visualized by Western blot and by immunofluorescent staining (red color), and the nuclei were visualized with DAPI (blue). The results are presented as merged images. The white scale bar represents 10 μm. c SNAIL silencing induces the expression of MYF5 mRNA in both undifferentiated RH30 cells and RH30 cells differentiated for 7 days in medium with 2% HS (qPCR, n = 3) d SNAIL silencing slightly induces the expression of MYF5 mRNA in both undifferentiated RH41 cells and RH41 cells differentiated for 7 days in medium with 2% HS (qPCR, n = 3). e Transient SNAIL silencing with siRNA in RH30 and RH41 ARMS cell lines slightly induces MYF5 expression. The cells were transfected with siRNA against SNAIL (siSNAIL) and a scrambled siRNA sequence (siRNA). Twenty-four hours after transfection, the cells were treated with differentiating medium containing 2% HS for the following 48 h. MYF5 levels were validated by qPCR; n = 3. f Transfection with pre-miR-30a-5p in RH30 cells induces MYF5 expression by downregulation of SNAIL protein. RH30 cells were transfected with the miRNA precursor pre-miR-30a-5p and pre-miR negative control (miR-neg-ctrl). Twenty-four hours after transfection, the cells were treated with differentiating medium containing 2% HS for the following 48 h. miR-30a-5p expression relative to U6 snRNA and MYF5, MYOD expression relative to GAPDH were validated by qPCR; n = 4. SNAIL protein level was validated by Western blot. The data represent the mean ± SEM. * p

    Article Snippet: Quantitative real-time PCR Gene expression was determined by qRT-PCR analysis using ABI PRISM 7300 Sequence Detection System or Quant Studio 7 Flex System (both from Applied Biosystems, Foster City, CA, USA), Blank qPCR Master Mix (EURx) and the indicated Taq-Man probes (Applied Biosystems): human: GAPDH (Hs99999905_m1), MYF5 (Hs00271574_m1), MYOD (Hs00159528_m1), MRF4 (Hs01547104_g1), MEF2A (Hs01050409_m1), MYOSTATIN (Hs00976237_m1), MYOGENIN (Hs01032275_m1), MYH2 (Hs00430042_m1), SNAI1 (Hs00195591_m1), HDAC1 (Hs02621185_s1) and HDAC2 (Hs00231032_m1).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining, Transfection, Sequencing, Negative Control

    Modification of ICE mES cells with Myf5, and efficient myogenesis in vitro

    Journal: Stem cells (Dayton, Ohio)

    Article Title: Inducible cassette exchange: a rapid and efficient system enabling conditional gene expression in embryonic stem and primary cells

    doi:

    Figure Lengend Snippet: Modification of ICE mES cells with Myf5, and efficient myogenesis in vitro

    Article Snippet: For Human probes: MYOD1 Hs00159528_m1, MYF5 Hs00271574_m1, MYOG Hs01072232_m1, DESMIN Hs00157258_m1, GAPDH Hs99999905-m1.

    Techniques: Modification, In Vitro

    Tbx6 Induced and Maintained a Mesoderm Program in Mouse Fibroblasts (A and B) qRT-PCR for Mesp1 expression after transduction of individual (A) or combinatorial (B) factors after 1 week (n = 3, independent experiments). Data were normalized to the values in MEFs. (C) Immunocytochemistry for Mesp1Cre-GFP and DAPI with quantification of the numbers of Mesp1Cre-GFP + colonies (n = 3, independent experiments). Tbx6 induced Mesp1Cre-GFP expression after 2 weeks of transduction. (D) Immunocytochemistry and quantification of the numbers of T + colonies (n = 3, independent experiments). Immunocytochemistry demonstrated that T protein was expressed in Tbx6transduced mouse embryonic fibroblast nuclei after 4 weeks. (F) FACS analyses showed that Tbx6transduced cells expressed cell-surface markers Flk1 and PDGFRα (n = 3, independent experiments). Time course of mRNA expression in MEFs transduced with Tbx6, as determined by qRT-PCR. Gene expression for mesoderm ( Mesp1 , T , Flk1 ), CPCs ( Isl1 , Nkx2.5 , Gata4 ), CMs ( Tnnt2 , Myh6 ), SMCs ( Myh11 ), ECs ( Pecam1 ), and skeletal muscle ( Pax3 , Pax7 , Myod1 , Myf5 ) genes were determined. Tbx6-transduced cells expressed mesoderm genes, but not those associated with cardiovascular differentiation and skeletal muscle genes. Data were normalized to the values on day 0. All data are presented as mean ± SD. **p

    Journal: Cell stem cell

    Article Title: Tbx6 Induces Nascent Mesoderm from Pluripotent Stem Cells and Temporally Controls Cardiac versus Somite Lineage Diversification

    doi: 10.1016/j.stem.2018.07.001

    Figure Lengend Snippet: Tbx6 Induced and Maintained a Mesoderm Program in Mouse Fibroblasts (A and B) qRT-PCR for Mesp1 expression after transduction of individual (A) or combinatorial (B) factors after 1 week (n = 3, independent experiments). Data were normalized to the values in MEFs. (C) Immunocytochemistry for Mesp1Cre-GFP and DAPI with quantification of the numbers of Mesp1Cre-GFP + colonies (n = 3, independent experiments). Tbx6 induced Mesp1Cre-GFP expression after 2 weeks of transduction. (D) Immunocytochemistry and quantification of the numbers of T + colonies (n = 3, independent experiments). Immunocytochemistry demonstrated that T protein was expressed in Tbx6transduced mouse embryonic fibroblast nuclei after 4 weeks. (F) FACS analyses showed that Tbx6transduced cells expressed cell-surface markers Flk1 and PDGFRα (n = 3, independent experiments). Time course of mRNA expression in MEFs transduced with Tbx6, as determined by qRT-PCR. Gene expression for mesoderm ( Mesp1 , T , Flk1 ), CPCs ( Isl1 , Nkx2.5 , Gata4 ), CMs ( Tnnt2 , Myh6 ), SMCs ( Myh11 ), ECs ( Pecam1 ), and skeletal muscle ( Pax3 , Pax7 , Myod1 , Myf5 ) genes were determined. Tbx6-transduced cells expressed mesoderm genes, but not those associated with cardiovascular differentiation and skeletal muscle genes. Data were normalized to the values on day 0. All data are presented as mean ± SD. **p

    Article Snippet: Mouse gene expression assays used the following probes: Acta1 (Mm808218_g1), Actn2 (Mm00473657_m1), Bmp4 (Mm00432087_m1), Cdx2 (Mm01212280_m1), Col2a1 (Mm01309565_m1), Eomes (Mm01351985_m1), Flk1 (Mm01222431 m1), Gapdh (Mm99999915_g1), Gsc (Mm00650681_g1), Isl1 (Mm00517585_m1), Lhx1 (Mm01297482_m1), Meox1 (Mm00440285_m1), Mesp1 (Mm00801883_g1), Msgn1 (Mm00490407_s1), Msx1 (Mm00440330_m1), Myf5 (Mm00435125_m1), Myh3 (Mm01332463_m1), Myh6 (Mm00440354_m1), Myh11 (Mm00443013_m1), Myog (Mm00446194_m1), Nkx2.5 (Mm00657783_m1), Nodal ( Mm00443040_m1), Pax3 (Mm00435491_m1), Pax6 (Mm00443081_m1), Pax7 (Mm01354484_m1), Pdgfr a (Mm00440701_m1), Pecam1 (Mm 01242584_m1), Sox1 (Mm00486299_s1), Sox2 (Mm03053810_s1), Sox9 (Mm00448840_m1), Sox17 (Mm00488363_m1), T (Mm00436877_m1), Tbx6 (Mm01278677_m1), Tcf15 (Mm00493442_m1), Tnnt2 (Mm00441922_m1), and Wnt3 (Mm00437336_m1).

    Techniques: Quantitative RT-PCR, Expressing, Transduction, Immunocytochemistry, FACS

    BETi have a memory effect on DUX4 that is mediated by HDACs. a – c BETi cause sustained DUX4 repression. a Experimental timeline. Subconfluent MB200 FSHD2 myoblasts were treated with 1 μM of the BETi I-BET762 (I-BET) on day 0 (D0) for 8, 24, 48, or 72 h and gene expression analyzed on day 3 (D3). b DUX4 target gene mRNA levels after treatment as in a . c Expression of the myoblast lineage genes MYOD1 and MYF5 and the epigenetic modifier SMCHD1 after treatment as in a . d – e HDACi block the BETi-mediated memory effect. d Levels of the DUX4 target gene ZSCAN4 in MB200 FSHD2 myoblasts that were treated with the indicated compounds (DMSO control, 1 μM I-BET762, 2.5 μM MS-275, 2.5 μM MGCD0103) for 24 h and then cultured in fresh media for an additional 48 h before harvest. e ZSCAN4 expression after 72 h of continuous exposure to the indicated compounds (DMSO control, 1 μM I-BET762, 2 μM RGFP109) in MB200 FSHD2 myoblasts. Error bars indicate the standard deviation from the mean of three biological replicates. p values were calculated using a one-way analysis of variance with Dunnett’s post test. * p

    Journal: Skeletal Muscle

    Article Title: BET bromodomain inhibitors and agonists of the beta-2 adrenergic receptor identified in screens for compounds that inhibit DUX4 expression in FSHD muscle cells

    doi: 10.1186/s13395-017-0134-x

    Figure Lengend Snippet: BETi have a memory effect on DUX4 that is mediated by HDACs. a – c BETi cause sustained DUX4 repression. a Experimental timeline. Subconfluent MB200 FSHD2 myoblasts were treated with 1 μM of the BETi I-BET762 (I-BET) on day 0 (D0) for 8, 24, 48, or 72 h and gene expression analyzed on day 3 (D3). b DUX4 target gene mRNA levels after treatment as in a . c Expression of the myoblast lineage genes MYOD1 and MYF5 and the epigenetic modifier SMCHD1 after treatment as in a . d – e HDACi block the BETi-mediated memory effect. d Levels of the DUX4 target gene ZSCAN4 in MB200 FSHD2 myoblasts that were treated with the indicated compounds (DMSO control, 1 μM I-BET762, 2.5 μM MS-275, 2.5 μM MGCD0103) for 24 h and then cultured in fresh media for an additional 48 h before harvest. e ZSCAN4 expression after 72 h of continuous exposure to the indicated compounds (DMSO control, 1 μM I-BET762, 2 μM RGFP109) in MB200 FSHD2 myoblasts. Error bars indicate the standard deviation from the mean of three biological replicates. p values were calculated using a one-way analysis of variance with Dunnett’s post test. * p

    Article Snippet: TaqMan gene expression assay ID numbers BRD2, Hs01121986_g1; BRD3, Hs00201284_m1; BRD4, Hs04188087_m1; BRDT, Hs00976114_m1; MBD3L2, Hs00544743_m1; MYF5, Hs00929416_g1; MYH2, Hs00430042_m1; MYOD1, Hs00159528_m1; MYOG, Hs01072232_m1; RPL13A, Hs04194366_g1; RPL30, Hs00265497_m1; SMCHD1, Hs00826906_m1; TRIM43, Hs00299174_m1; ZSCAN4, Hs00537549_m1; DUX4, primers GCCGGCCCAGGTACCA and CAGCGAGCTCCCTTGCA with probe 6FAMCAGTGCGCACCCCGMGBNFQ.

    Techniques: Expressing, Blocking Assay, Mass Spectrometry, Cell Culture, Standard Deviation