myf5 Thermo Fisher Search Results


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  • 89
    Thermo Fisher anti myf5 primary antibody
    Ischemia-induced satellite-cell activation is impaired in young p75KO mice. Representative ×63 confocal microscopy images for triple immunostaining with satellite-cell marker: <t>Myf5</t> (red), laminin (green) to visualize the basement membrane, and
    Anti Myf5 Primary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: 93/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    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: 85/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gene exp myf5 hs00271574 m1
    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 Hs00271574 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 82/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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: 88/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti myf 5 polyclonal antibody
    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
    Anti Myf 5 Polyclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher gene exp myod1 mm00440387 m1
    Effect of homeodomain substitution mutants on myogenesis. (A) RT-qPCR analyses for <t>MyoD</t> and Myf5 expression in the cells induced with 250 ng ml −1 doxycycline in proliferation medium for 8 h. Gene expression was normalized to GAPDH expression and presented as fold difference compared with the control samples ( n =4). Error bar represents mean±s.e.m., one-way ANOVA. (B) Immunofluorescence for MHC (red), DUX4 (green) and nuclear staining with DAPI (blue) in cells induced with 250 ng ml −1 doxycycline for 4 days during myogenic differentiation. (C) Fusion index in cells induced with 25 and 250 ng ml −1 doxycycline for 4 days during differentiation ( n =6). Data is presented as fold difference of the mean±s.e.m., two-way ANOVA; *** P
    Gene Exp Myod1 Mm00440387 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 153 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gene exp bad hs00188930 m1
    Effect of homeodomain substitution mutants on myogenesis. (A) RT-qPCR analyses for <t>MyoD</t> and Myf5 expression in the cells induced with 250 ng ml −1 doxycycline in proliferation medium for 8 h. Gene expression was normalized to GAPDH expression and presented as fold difference compared with the control samples ( n =4). Error bar represents mean±s.e.m., one-way ANOVA. (B) Immunofluorescence for MHC (red), DUX4 (green) and nuclear staining with DAPI (blue) in cells induced with 250 ng ml −1 doxycycline for 4 days during myogenic differentiation. (C) Fusion index in cells induced with 25 and 250 ng ml −1 doxycycline for 4 days during differentiation ( n =6). Data is presented as fold difference of the mean±s.e.m., two-way ANOVA; *** P
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    Thermo Fisher gene exp zscan4 hs00537549 m1
    DUX4 interacts with p300/CBP. ( A ) Schematic diagram of the lentiviral constructs carrying doxycycline-inducible DUX4 and rtTA. sgTRE: second generation tet-response element for doxycycline-induced expression. ( B ) Immunoblot showing doxycycline dose-dependent inducible expression of DUX4-flag in LHCN-M2 cells. ( C ) Time course of induction of DUX4 downstream target genes, FRG2 and <t>ZSCAN4</t> . ( D ) Cell death of immortalized human LHCN-M2 myoblasts 48 hours after DUX4 induction with 250 ng/ml doxycycline. ( E ) Silver stained SDS-PAGE gel of DUX4-flag associated proteins, 6 h after 250 ng/ml doxycycline treatment. ( F ) Immunoblots of DUX4-associated factors, p300 and CBP, in immunoprecipitates with Flag-DUX4. ( G ) Direct interaction of DUX4 with p300. Left panel: Coomassie staining of GST and GST-DUX4 proteins purified from E. coli . Right panel: immunoblot for recombinant p300 after GST-pull down, showing interaction with GST-DUX4 but not GST alone.
    Gene Exp Zscan4 Hs00537549 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MYF5 Ala88 Polyclonal Antibody for Western Blot
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    Image Search Results


    Ischemia-induced satellite-cell activation is impaired in young p75KO mice. Representative ×63 confocal microscopy images for triple immunostaining with satellite-cell marker: Myf5 (red), laminin (green) to visualize the basement membrane, and

    Journal: The FASEB Journal

    Article Title: Genetic deletion of TNFR2 augments inflammatory response and blunts satellite-cell-mediated recovery response in a hind limb ischemia model

    doi: 10.1096/fj.14-249813

    Figure Lengend Snippet: Ischemia-induced satellite-cell activation is impaired in young p75KO mice. Representative ×63 confocal microscopy images for triple immunostaining with satellite-cell marker: Myf5 (red), laminin (green) to visualize the basement membrane, and

    Article Snippet: Muscle tissue sections were sequentially stained, first with anti-Myf5 primary antibody followed by Alexa-Fluor 555 goat anti-rabbit secondary antibody (Life Technologies), then with anti-laminin primary antibody followed by Alexa Fluor 488 goat anti-rabbit secondary antibody (Life Technologies).

    Techniques: Activation Assay, Mouse Assay, Confocal Microscopy, Triple Immunostaining, Marker

    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

    Journal: Stem Cell Research & Therapy

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

    doi: 10.1186/s13287-018-1096-6

    Figure Lengend 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

    Article Snippet: Probes used for evaluating the degree of differentiation in myoblasts included: MYF5 (Applied Biosystems; #Rn01502778; #Mm00435125), MYH1 (Applied Biosystems; #Rn01751056; Mm01332489), and MYH2 (Applied Biosystems; #Rn01470656; Mm01332564).

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

    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

    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

    Effect of homeodomain substitution mutants on myogenesis. (A) RT-qPCR analyses for MyoD and Myf5 expression in the cells induced with 250 ng ml −1 doxycycline in proliferation medium for 8 h. Gene expression was normalized to GAPDH expression and presented as fold difference compared with the control samples ( n =4). Error bar represents mean±s.e.m., one-way ANOVA. (B) Immunofluorescence for MHC (red), DUX4 (green) and nuclear staining with DAPI (blue) in cells induced with 250 ng ml −1 doxycycline for 4 days during myogenic differentiation. (C) Fusion index in cells induced with 25 and 250 ng ml −1 doxycycline for 4 days during differentiation ( n =6). Data is presented as fold difference of the mean±s.e.m., two-way ANOVA; *** P

    Journal: Journal of Cell Science

    Article Title: The DUX4 homeodomains mediate inhibition of myogenesis and are functionally exchangeable with the Pax7 homeodomain

    doi: 10.1242/jcs.205427

    Figure Lengend Snippet: Effect of homeodomain substitution mutants on myogenesis. (A) RT-qPCR analyses for MyoD and Myf5 expression in the cells induced with 250 ng ml −1 doxycycline in proliferation medium for 8 h. Gene expression was normalized to GAPDH expression and presented as fold difference compared with the control samples ( n =4). Error bar represents mean±s.e.m., one-way ANOVA. (B) Immunofluorescence for MHC (red), DUX4 (green) and nuclear staining with DAPI (blue) in cells induced with 250 ng ml −1 doxycycline for 4 days during myogenic differentiation. (C) Fusion index in cells induced with 25 and 250 ng ml −1 doxycycline for 4 days during differentiation ( n =6). Data is presented as fold difference of the mean±s.e.m., two-way ANOVA; *** P

    Article Snippet: PCR was performed by using TaqMan Real-Time PCR premixture and premix probes (Myod1 Mm00440387_m1, Myf5 Mm00435125_m1, human MYOD1 Hs00159528_m1) on a 7900 HT real-time PCR System (Applied Biosystems, Carlsbad, CA).

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

    Effect of DUX4 deletions on myogenesis during proliferation and terminal differentiation. (A) RT-qPCR analyses for MyoD and Myf5 expression in cell lines expressing various deletion constructs induced with 250 ng ml −1 doxycycline for 6 h in proliferation medium and compared with uninduced controls ( n =4). Gene expression was normalized to the levels of GAPDH. Data are presented as mean±s.e.m., one-way ANOVA. (B) Analysis of myogenic differentiation of the deletion constructs by immunofluorescence for MHC (red). Nuclei were visualized by DAPI staining (blue). Doxycycline (250 ng ml −1 ) was added for 4 days while cells were cultured in differentiation media. (C) Fusion index analyses for evaluation of levels of myogenic differentiation. Cell lines were differentiated with 25 and 250 ng ml −1 doxycycline for 4 days ( n =6). Values of induced cells were normalized to the uninduced group. Data are presented as fold difference; mean±s.e.m., two-way ANOVA. (D) Immunostaining for MHC (red), DUX4 (green) and DAPI (blue staining of the nuclei) in iC2C12-DUX4(1–399) at day 4 of differentiation. Cells were induced with 25 ng ml −1 doxycycline. Note that all of the cells in the doxycycline-treated group expressed DUX4(1–399). (E) Immunostaining for MyoD (red) and counterstaining of nuclei with DAPI (blue) in iC2C12-DUX4(1–399) at day 4 of differentiation. Note that staining for MyoD is decreased in cells induced with 25 ng ml −1 doxycycline. * P

    Journal: Journal of Cell Science

    Article Title: The DUX4 homeodomains mediate inhibition of myogenesis and are functionally exchangeable with the Pax7 homeodomain

    doi: 10.1242/jcs.205427

    Figure Lengend Snippet: Effect of DUX4 deletions on myogenesis during proliferation and terminal differentiation. (A) RT-qPCR analyses for MyoD and Myf5 expression in cell lines expressing various deletion constructs induced with 250 ng ml −1 doxycycline for 6 h in proliferation medium and compared with uninduced controls ( n =4). Gene expression was normalized to the levels of GAPDH. Data are presented as mean±s.e.m., one-way ANOVA. (B) Analysis of myogenic differentiation of the deletion constructs by immunofluorescence for MHC (red). Nuclei were visualized by DAPI staining (blue). Doxycycline (250 ng ml −1 ) was added for 4 days while cells were cultured in differentiation media. (C) Fusion index analyses for evaluation of levels of myogenic differentiation. Cell lines were differentiated with 25 and 250 ng ml −1 doxycycline for 4 days ( n =6). Values of induced cells were normalized to the uninduced group. Data are presented as fold difference; mean±s.e.m., two-way ANOVA. (D) Immunostaining for MHC (red), DUX4 (green) and DAPI (blue staining of the nuclei) in iC2C12-DUX4(1–399) at day 4 of differentiation. Cells were induced with 25 ng ml −1 doxycycline. Note that all of the cells in the doxycycline-treated group expressed DUX4(1–399). (E) Immunostaining for MyoD (red) and counterstaining of nuclei with DAPI (blue) in iC2C12-DUX4(1–399) at day 4 of differentiation. Note that staining for MyoD is decreased in cells induced with 25 ng ml −1 doxycycline. * P

    Article Snippet: PCR was performed by using TaqMan Real-Time PCR premixture and premix probes (Myod1 Mm00440387_m1, Myf5 Mm00435125_m1, human MYOD1 Hs00159528_m1) on a 7900 HT real-time PCR System (Applied Biosystems, Carlsbad, CA).

    Techniques: Quantitative RT-PCR, Expressing, Construct, Immunofluorescence, Staining, Cell Culture, Immunostaining

    MYOD overexpression suppresses DUX4-dependent inhibition of differentiation. (A) FACS analyses of iC2C12-DUX4 (1–377) infected with retrovirus carrying GFP or human MYOD-ires-GPP expression vectors. (B) RT-qPCR for human MYOD (MYOD) and endogenous MyoD expression in DUX4 C-terminal deletion, i.e. DUX4(1–377), expressing cell lines transduced with either GFP control (labeled ‘ GFP’) or human MYOD1 (labeled ‘ MYOD’). Human MYOD1 is shown relative to GAPDH control. Endogenous murine MyoD1 is shown relative to its expression in the absence of Dox. Cells were induced with 250 ng ml −1 doxycycline for 14 h ( n =4). Data are presented as fold difference compared with control (uninduced cells), mean±s.e.m.; one-way ANOVA. Note that only the DUX4(1–377) MYOD cell line expressed MYOD. (C) Immunostaining for MHC (red) and DAPI (blue, nuclei) in differentiating cells at day 4 post-induction of differentiation (doxycycline 250 ng ml −1 , or control, no dox). Scale bars: 50 µm. (D) Fusion index analysis of the level of differentiation in cells 4 days after differentiation was induced in the presence of 250 ng ml −1 doxycycline ( n =6). Fusion index represents the ratio of nuclei within myotubes to the total number of nuclei; mean±s.e.m.; two-way ANOVA. **** P

    Journal: Journal of Cell Science

    Article Title: The DUX4 homeodomains mediate inhibition of myogenesis and are functionally exchangeable with the Pax7 homeodomain

    doi: 10.1242/jcs.205427

    Figure Lengend Snippet: MYOD overexpression suppresses DUX4-dependent inhibition of differentiation. (A) FACS analyses of iC2C12-DUX4 (1–377) infected with retrovirus carrying GFP or human MYOD-ires-GPP expression vectors. (B) RT-qPCR for human MYOD (MYOD) and endogenous MyoD expression in DUX4 C-terminal deletion, i.e. DUX4(1–377), expressing cell lines transduced with either GFP control (labeled ‘ GFP’) or human MYOD1 (labeled ‘ MYOD’). Human MYOD1 is shown relative to GAPDH control. Endogenous murine MyoD1 is shown relative to its expression in the absence of Dox. Cells were induced with 250 ng ml −1 doxycycline for 14 h ( n =4). Data are presented as fold difference compared with control (uninduced cells), mean±s.e.m.; one-way ANOVA. Note that only the DUX4(1–377) MYOD cell line expressed MYOD. (C) Immunostaining for MHC (red) and DAPI (blue, nuclei) in differentiating cells at day 4 post-induction of differentiation (doxycycline 250 ng ml −1 , or control, no dox). Scale bars: 50 µm. (D) Fusion index analysis of the level of differentiation in cells 4 days after differentiation was induced in the presence of 250 ng ml −1 doxycycline ( n =6). Fusion index represents the ratio of nuclei within myotubes to the total number of nuclei; mean±s.e.m.; two-way ANOVA. **** P

    Article Snippet: PCR was performed by using TaqMan Real-Time PCR premixture and premix probes (Myod1 Mm00440387_m1, Myf5 Mm00435125_m1, human MYOD1 Hs00159528_m1) on a 7900 HT real-time PCR System (Applied Biosystems, Carlsbad, CA).

    Techniques: Over Expression, Inhibition, FACS, Infection, Expressing, Quantitative RT-PCR, Transduction, Labeling, Immunostaining

    DUX4 interacts with p300/CBP. ( A ) Schematic diagram of the lentiviral constructs carrying doxycycline-inducible DUX4 and rtTA. sgTRE: second generation tet-response element for doxycycline-induced expression. ( B ) Immunoblot showing doxycycline dose-dependent inducible expression of DUX4-flag in LHCN-M2 cells. ( C ) Time course of induction of DUX4 downstream target genes, FRG2 and ZSCAN4 . ( D ) Cell death of immortalized human LHCN-M2 myoblasts 48 hours after DUX4 induction with 250 ng/ml doxycycline. ( E ) Silver stained SDS-PAGE gel of DUX4-flag associated proteins, 6 h after 250 ng/ml doxycycline treatment. ( F ) Immunoblots of DUX4-associated factors, p300 and CBP, in immunoprecipitates with Flag-DUX4. ( G ) Direct interaction of DUX4 with p300. Left panel: Coomassie staining of GST and GST-DUX4 proteins purified from E. coli . Right panel: immunoblot for recombinant p300 after GST-pull down, showing interaction with GST-DUX4 but not GST alone.

    Journal: Nucleic Acids Research

    Article Title: DUX4 recruits p300/CBP through its C-terminus and induces global H3K27 acetylation changes

    doi: 10.1093/nar/gkw141

    Figure Lengend Snippet: DUX4 interacts with p300/CBP. ( A ) Schematic diagram of the lentiviral constructs carrying doxycycline-inducible DUX4 and rtTA. sgTRE: second generation tet-response element for doxycycline-induced expression. ( B ) Immunoblot showing doxycycline dose-dependent inducible expression of DUX4-flag in LHCN-M2 cells. ( C ) Time course of induction of DUX4 downstream target genes, FRG2 and ZSCAN4 . ( D ) Cell death of immortalized human LHCN-M2 myoblasts 48 hours after DUX4 induction with 250 ng/ml doxycycline. ( E ) Silver stained SDS-PAGE gel of DUX4-flag associated proteins, 6 h after 250 ng/ml doxycycline treatment. ( F ) Immunoblots of DUX4-associated factors, p300 and CBP, in immunoprecipitates with Flag-DUX4. ( G ) Direct interaction of DUX4 with p300. Left panel: Coomassie staining of GST and GST-DUX4 proteins purified from E. coli . Right panel: immunoblot for recombinant p300 after GST-pull down, showing interaction with GST-DUX4 but not GST alone.

    Article Snippet: Reverse transcription (RT) was performed using the Vero cDNA synthesis kit (Thermo Scientific) and quantitative polymerase chain reactions (PCR) were performed using Premix Ex Taq™,ROX Plus (Clontech) and hydrolysis (Taqman) probes (ZSCAN4; Hs00537549_m1, MYF5; Hs00271574_m1, MYOD1; Hs00159528_m1, CCNA1; Hs00171105_m1, GAPDH; Hs99999905_m1) (Applied Biosystems).

    Techniques: Construct, Expressing, Staining, SDS Page, Western Blot, Purification, Recombinant

    p300 is recruited to the DUX4 binding site in ZSCAN4 . ( A ) Location of PCR amplicons in the ZSCAN4 gene used for ChIP analyses. The previously-identified DUX4-binding site is at +1430, in exon 2. ( B–G ) ChIP assays with various antibodies at the indicated ZSCAN4 loci (ZSCAN4-P: promoter region, ZSCAN4-E: exon 2 region). LHCN-M2 cells were treated with 250 ng/ml dox for 6 h to express DUX4 or DUX4-ΔC and subjected to ChIP analyses.% input of H3K18Ac, H3K27Ac, and H3K4me3 were normalized with% input of H3 ( n = 3, error bars represent SEM). Note that both DUX4 and DUX4ΔC are recruited to the DUX4 site in exon 2, but p300 is not recruited by DUX4ΔC, and acetylation of H3K18 is not increased by DUX4ΔC.

    Journal: Nucleic Acids Research

    Article Title: DUX4 recruits p300/CBP through its C-terminus and induces global H3K27 acetylation changes

    doi: 10.1093/nar/gkw141

    Figure Lengend Snippet: p300 is recruited to the DUX4 binding site in ZSCAN4 . ( A ) Location of PCR amplicons in the ZSCAN4 gene used for ChIP analyses. The previously-identified DUX4-binding site is at +1430, in exon 2. ( B–G ) ChIP assays with various antibodies at the indicated ZSCAN4 loci (ZSCAN4-P: promoter region, ZSCAN4-E: exon 2 region). LHCN-M2 cells were treated with 250 ng/ml dox for 6 h to express DUX4 or DUX4-ΔC and subjected to ChIP analyses.% input of H3K18Ac, H3K27Ac, and H3K4me3 were normalized with% input of H3 ( n = 3, error bars represent SEM). Note that both DUX4 and DUX4ΔC are recruited to the DUX4 site in exon 2, but p300 is not recruited by DUX4ΔC, and acetylation of H3K18 is not increased by DUX4ΔC.

    Article Snippet: Reverse transcription (RT) was performed using the Vero cDNA synthesis kit (Thermo Scientific) and quantitative polymerase chain reactions (PCR) were performed using Premix Ex Taq™,ROX Plus (Clontech) and hydrolysis (Taqman) probes (ZSCAN4; Hs00537549_m1, MYF5; Hs00271574_m1, MYOD1; Hs00159528_m1, CCNA1; Hs00171105_m1, GAPDH; Hs99999905_m1) (Applied Biosystems).

    Techniques: Binding Assay, Polymerase Chain Reaction, Chromatin Immunoprecipitation

    Genome wide changes in histone H3 and H3K27ac occupancy by DUX4 in LHCN-M2-iDUX4 cells. ( A ) Pan-histone H3 and H3K27Ac changes at ZSCAN4 after DUX4 expression. Red and black ovals represent the TAA T TCAATCA and TAA A TCAATCA DUX4 homeodomain binding motifs respectively. A strong DUX4 peak, shown in blue, is observed at these motifs proximal to an alternate promoter for ZSCAN4 (Ensembl Transcript ENST00000612521); pan-Histone H3 signal before (–) and after (+) doxycycline treatment are shown in green; H3K27Ac signals before and after doxycycline are shown in red. Note the loss of histone H3 signal and the appearance of H3K27Ac peaks that form adjacent to the site of DUX4 binding. ( B ) Genome wide changes in chromatin upon DUX4 induction. The distribution in the number of sequence reads in the H3K27ac datasets that map to within 1 kb of 31 042 Dux4 peak summits is shown as a violin plot in absence or presence of doxycycline. ( C ) The ChIP signals 4 kb upstream and downstream of the peak summits were summed across all Dux4 binding sites, normalized to the mapped sequencing depth and log2 transformed. The upper panel contains reads for the input (yellow) and Flag-DUX4 ChIP (blue) samples. The lower panel contains the H3K27Ac ChIP signals that were observed before (orange) and after (blue) doxycycline treatment. ( D ) The regions bound by DUX4 used in panels (B) and (C) were separated into 12 912 peaks that overlap DNase hypersensitivity sites (left panels) and 18 130 peaks that do not (right panels). ChIP signals are shown for input, Flag-DUX4 ChIP, histone H3 ChIP, H3K27Ac ChIP and H3K4me3 ChIP. Data are colored as in panel C: input = yellow, absence of doxycycline = orange, presence of doxycycline = blue. Note that a decrease is observed in the histone H3 signal upon DUX4 induction in the DNase in-accessible regions and that a similar decrease preexisted in the DNase accessible DUX4 binding regions. A saddle-shaped distribution of H3K27Ac ChIP signal is observed at DUX4 peaks at DNase accessible regions and upon DUX4 induction in DNase in-accessible regions.

    Journal: Nucleic Acids Research

    Article Title: DUX4 recruits p300/CBP through its C-terminus and induces global H3K27 acetylation changes

    doi: 10.1093/nar/gkw141

    Figure Lengend Snippet: Genome wide changes in histone H3 and H3K27ac occupancy by DUX4 in LHCN-M2-iDUX4 cells. ( A ) Pan-histone H3 and H3K27Ac changes at ZSCAN4 after DUX4 expression. Red and black ovals represent the TAA T TCAATCA and TAA A TCAATCA DUX4 homeodomain binding motifs respectively. A strong DUX4 peak, shown in blue, is observed at these motifs proximal to an alternate promoter for ZSCAN4 (Ensembl Transcript ENST00000612521); pan-Histone H3 signal before (–) and after (+) doxycycline treatment are shown in green; H3K27Ac signals before and after doxycycline are shown in red. Note the loss of histone H3 signal and the appearance of H3K27Ac peaks that form adjacent to the site of DUX4 binding. ( B ) Genome wide changes in chromatin upon DUX4 induction. The distribution in the number of sequence reads in the H3K27ac datasets that map to within 1 kb of 31 042 Dux4 peak summits is shown as a violin plot in absence or presence of doxycycline. ( C ) The ChIP signals 4 kb upstream and downstream of the peak summits were summed across all Dux4 binding sites, normalized to the mapped sequencing depth and log2 transformed. The upper panel contains reads for the input (yellow) and Flag-DUX4 ChIP (blue) samples. The lower panel contains the H3K27Ac ChIP signals that were observed before (orange) and after (blue) doxycycline treatment. ( D ) The regions bound by DUX4 used in panels (B) and (C) were separated into 12 912 peaks that overlap DNase hypersensitivity sites (left panels) and 18 130 peaks that do not (right panels). ChIP signals are shown for input, Flag-DUX4 ChIP, histone H3 ChIP, H3K27Ac ChIP and H3K4me3 ChIP. Data are colored as in panel C: input = yellow, absence of doxycycline = orange, presence of doxycycline = blue. Note that a decrease is observed in the histone H3 signal upon DUX4 induction in the DNase in-accessible regions and that a similar decrease preexisted in the DNase accessible DUX4 binding regions. A saddle-shaped distribution of H3K27Ac ChIP signal is observed at DUX4 peaks at DNase accessible regions and upon DUX4 induction in DNase in-accessible regions.

    Article Snippet: Reverse transcription (RT) was performed using the Vero cDNA synthesis kit (Thermo Scientific) and quantitative polymerase chain reactions (PCR) were performed using Premix Ex Taq™,ROX Plus (Clontech) and hydrolysis (Taqman) probes (ZSCAN4; Hs00537549_m1, MYF5; Hs00271574_m1, MYOD1; Hs00159528_m1, CCNA1; Hs00171105_m1, GAPDH; Hs99999905_m1) (Applied Biosystems).

    Techniques: Genome Wide, Expressing, Binding Assay, Sequencing, Chromatin Immunoprecipitation, Transformation Assay

    The central unstructured domain of DUX4 is dispensable for transcriptional activation and cytotoxicity. ( A ) Scheme of DUX4 deletion mutants. HD: homeodomain. GFP was added to visualize DUX4 fragments. The number of DUX4 amino acids in the N-terminal and C-terminal parts of each construct are indicated. ( B ) RT-qPCR analysis of RNA of the target gene ZSCAN4 after transient transfection of each construct. Protein expression levels of DUX4 deletion mutants are shown by immune blot, below. ( C ) Viability of C2C12 cells expressing different levels of each DUX4 deletion mutant. All constructs were targeted into the same genomic inducible locus. Cell viability (ATP content) was measured 24 h after treatment with doxycycline.

    Journal: Nucleic Acids Research

    Article Title: DUX4 recruits p300/CBP through its C-terminus and induces global H3K27 acetylation changes

    doi: 10.1093/nar/gkw141

    Figure Lengend Snippet: The central unstructured domain of DUX4 is dispensable for transcriptional activation and cytotoxicity. ( A ) Scheme of DUX4 deletion mutants. HD: homeodomain. GFP was added to visualize DUX4 fragments. The number of DUX4 amino acids in the N-terminal and C-terminal parts of each construct are indicated. ( B ) RT-qPCR analysis of RNA of the target gene ZSCAN4 after transient transfection of each construct. Protein expression levels of DUX4 deletion mutants are shown by immune blot, below. ( C ) Viability of C2C12 cells expressing different levels of each DUX4 deletion mutant. All constructs were targeted into the same genomic inducible locus. Cell viability (ATP content) was measured 24 h after treatment with doxycycline.

    Article Snippet: Reverse transcription (RT) was performed using the Vero cDNA synthesis kit (Thermo Scientific) and quantitative polymerase chain reactions (PCR) were performed using Premix Ex Taq™,ROX Plus (Clontech) and hydrolysis (Taqman) probes (ZSCAN4; Hs00537549_m1, MYF5; Hs00271574_m1, MYOD1; Hs00159528_m1, CCNA1; Hs00171105_m1, GAPDH; Hs99999905_m1) (Applied Biosystems).

    Techniques: Activation Assay, Construct, Quantitative RT-PCR, Transfection, Expressing, Mutagenesis

    Dominant negative activity of the p300 interacting C-terminal domain of DUX4. ( A ) Schematic of DUX4 expression constructs. 293T cells modified to express full-length DUX4 driven from the dox-inducible sgTRE promoter, shown above, were transfected with transient expression constructs, shown below. The number of C-terminal DUX4 amino acids is indicated. sgTRE: second generation tet-response element for dox-induced expression; CMV: CMV promoter for high level constitutive expression; NLS: SV40-nuclear localization signal; HD: homeodomain. GFP was added to stabilize DUX4 fragments. NLS was added to deliver DUX4 fragment in the nucleus. ( B ) DUX4 mediated ZSCAN4 mRNA expression. Cells were transfected, 24 h later 250 ng/ml dox was applied, and mRNA extracted 6 hours after that ( n = 3, error bars represent SEM). ( C ) Activity of a cotransfected DUX4-luciferase reporter. 293T cells were co-transfected with both full length DUX4 and empty vector (EV) or the tested fragments, together with DUX4 luciferase and Renilla control reporters ( n = 3, error bars represent SEM). ( D ) Western blots of DUX4 immunoprecipitations performed in the presence of GFP-NLS control or GFP-NLS-DUX4-C-terminal 98 amino acids. Interaction of full length DUX4 with p300 is inhibited by overexpression of the C-terminus of DUX4.

    Journal: Nucleic Acids Research

    Article Title: DUX4 recruits p300/CBP through its C-terminus and induces global H3K27 acetylation changes

    doi: 10.1093/nar/gkw141

    Figure Lengend Snippet: Dominant negative activity of the p300 interacting C-terminal domain of DUX4. ( A ) Schematic of DUX4 expression constructs. 293T cells modified to express full-length DUX4 driven from the dox-inducible sgTRE promoter, shown above, were transfected with transient expression constructs, shown below. The number of C-terminal DUX4 amino acids is indicated. sgTRE: second generation tet-response element for dox-induced expression; CMV: CMV promoter for high level constitutive expression; NLS: SV40-nuclear localization signal; HD: homeodomain. GFP was added to stabilize DUX4 fragments. NLS was added to deliver DUX4 fragment in the nucleus. ( B ) DUX4 mediated ZSCAN4 mRNA expression. Cells were transfected, 24 h later 250 ng/ml dox was applied, and mRNA extracted 6 hours after that ( n = 3, error bars represent SEM). ( C ) Activity of a cotransfected DUX4-luciferase reporter. 293T cells were co-transfected with both full length DUX4 and empty vector (EV) or the tested fragments, together with DUX4 luciferase and Renilla control reporters ( n = 3, error bars represent SEM). ( D ) Western blots of DUX4 immunoprecipitations performed in the presence of GFP-NLS control or GFP-NLS-DUX4-C-terminal 98 amino acids. Interaction of full length DUX4 with p300 is inhibited by overexpression of the C-terminus of DUX4.

    Article Snippet: Reverse transcription (RT) was performed using the Vero cDNA synthesis kit (Thermo Scientific) and quantitative polymerase chain reactions (PCR) were performed using Premix Ex Taq™,ROX Plus (Clontech) and hydrolysis (Taqman) probes (ZSCAN4; Hs00537549_m1, MYF5; Hs00271574_m1, MYOD1; Hs00159528_m1, CCNA1; Hs00171105_m1, GAPDH; Hs99999905_m1) (Applied Biosystems).

    Techniques: Dominant Negative Mutation, Activity Assay, Expressing, Construct, Modification, Transfection, Luciferase, Plasmid Preparation, Western Blot, Over Expression