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  • 99
    FUJIFILM β glycerophosphate
    β Glycerophosphate, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery non targeting pool d 001810
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    Horizon Discovery non targeting control sirnas
    Cdon expression rescues fusion defects caused by Ash1L knockdown. a Immunofluorescence for myosin heavy chain and nuclear staining in C2C12 cells treated with two consecutive rounds of transient transfections: non-targeting (NS) or Ash1L <t>siRNAs</t> ( Ash1L KD), and empty vector (EV) or Cdon- expressing vector ( Cdon OE), before inducing differentiation for 3 days (left). Scale bar, 100 µm. b RT-qPCR analysis of Ash1L and Cdon expression in C2C12 cells <t>transfected</t> with: empty vector plus non-targeting siRNAs (EV + NS), empty vector plus Ash1L siRNAs (EV + Ash1L KD), Cdon- expressing vector plus non-targeting siRNAs ( Cdon OE + NS), and Cdon- expressing vector + Ash1L siRNAs ( Cdon OE + Ash1L KD). c Fusion index and nuclei distribution for each condition were calculated as described for Fig. 3 . Unpaired two-tailed t test. Confidence intervals 95%. n = 3. Source data are provided as a Source Data file. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. NS not significant
    Non Targeting Control Sirnas, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dmem
    Cdon expression rescues fusion defects caused by Ash1L knockdown. a Immunofluorescence for myosin heavy chain and nuclear staining in C2C12 cells treated with two consecutive rounds of transient transfections: non-targeting (NS) or Ash1L <t>siRNAs</t> ( Ash1L KD), and empty vector (EV) or Cdon- expressing vector ( Cdon OE), before inducing differentiation for 3 days (left). Scale bar, 100 µm. b RT-qPCR analysis of Ash1L and Cdon expression in C2C12 cells <t>transfected</t> with: empty vector plus non-targeting siRNAs (EV + NS), empty vector plus Ash1L siRNAs (EV + Ash1L KD), Cdon- expressing vector plus non-targeting siRNAs ( Cdon OE + NS), and Cdon- expressing vector + Ash1L siRNAs ( Cdon OE + Ash1L KD). c Fusion index and nuclei distribution for each condition were calculated as described for Fig. 3 . Unpaired two-tailed t test. Confidence intervals 95%. n = 3. Source data are provided as a Source Data file. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. NS not significant
    Dmem, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Cdon expression rescues fusion defects caused by Ash1L knockdown. a Immunofluorescence for myosin heavy chain and nuclear staining in C2C12 cells treated with two consecutive rounds of transient transfections: non-targeting (NS) or Ash1L siRNAs ( Ash1L KD), and empty vector (EV) or Cdon- expressing vector ( Cdon OE), before inducing differentiation for 3 days (left). Scale bar, 100 µm. b RT-qPCR analysis of Ash1L and Cdon expression in C2C12 cells transfected with: empty vector plus non-targeting siRNAs (EV + NS), empty vector plus Ash1L siRNAs (EV + Ash1L KD), Cdon- expressing vector plus non-targeting siRNAs ( Cdon OE + NS), and Cdon- expressing vector + Ash1L siRNAs ( Cdon OE + Ash1L KD). c Fusion index and nuclei distribution for each condition were calculated as described for Fig. 3 . Unpaired two-tailed t test. Confidence intervals 95%. n = 3. Source data are provided as a Source Data file. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. NS not significant

    Journal: Nature Communications

    Article Title: The Trithorax protein Ash1L promotes myoblast fusion by activating Cdon expression

    doi: 10.1038/s41467-018-07313-8

    Figure Lengend Snippet: Cdon expression rescues fusion defects caused by Ash1L knockdown. a Immunofluorescence for myosin heavy chain and nuclear staining in C2C12 cells treated with two consecutive rounds of transient transfections: non-targeting (NS) or Ash1L siRNAs ( Ash1L KD), and empty vector (EV) or Cdon- expressing vector ( Cdon OE), before inducing differentiation for 3 days (left). Scale bar, 100 µm. b RT-qPCR analysis of Ash1L and Cdon expression in C2C12 cells transfected with: empty vector plus non-targeting siRNAs (EV + NS), empty vector plus Ash1L siRNAs (EV + Ash1L KD), Cdon- expressing vector plus non-targeting siRNAs ( Cdon OE + NS), and Cdon- expressing vector + Ash1L siRNAs ( Cdon OE + Ash1L KD). c Fusion index and nuclei distribution for each condition were calculated as described for Fig. 3 . Unpaired two-tailed t test. Confidence intervals 95%. n = 3. Source data are provided as a Source Data file. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. NS not significant

    Article Snippet: The day after, cells were transfected with small interfering RNAs (siRNAs) against Ash1L or non-targeting control siRNAs [SMARTpool: ON-TARGETplus ASH1L siRNA L-020460 (human), L-041891 (mouse) or Non-Targeting Pool D-001810; Dharmacon], using Lipofectamine 3000 reagent (Thermo Fisher Scientific) following the manufacturer’s instructions.

    Techniques: Expressing, Immunofluorescence, Staining, Transfection, Plasmid Preparation, Quantitative RT-PCR, Two Tailed Test

    The fusion gene Cdon is a direct target of Ash1L. Cdon expression positively correlates with Ash1L during muscle development ( a ) and regeneration ( b ). RT-qPCR analysis on muscle tissue from hindlimbs of mice from the embryonic stage E16.5 to adulthood and in tibialis anterior of wild-type adult mice, untreated (UNT), or 5 and 10 days after cardiotoxin (CTX) injection. Unpaired two-tailed t test. Confidence intervals 95%. n = 6 ( a ), n = 4 ( b ). c Cdon protein level during in vitro muscle differentiation and in proliferating myoblasts vs. confluent cells. Immunoblot of C2C12 cells at days 0 and 1, and densitometric analysis of Cdon signal relative to vinculin as housekeeping protein (left panel). Comparison between proliferating myoblasts (P) and confluent cells (C) (right panel). Paired two-tailed t test. Confidence intervals 95%. Data are the mean for three independent experiments. d RT-qPCR analysis comparing Cdon expression in the presence and absence of Ash1L in three different models: muscle tissue from hindlimbs of wild-type (WT) and Ash1L GT embryos at E18.5, C2C12 cells transfected with non-targeting (NS) or Ash1L siRNAs ( Ash1L KD), and human primary cells transfected with non-targeting or Ash1L siRNAs, and collected at day 1 of differentiation. Unpaired two-tailed t test. Confidence intervals 95%. n = 6 (muscle tissue), four independent experiments (C2C12 cells), and three independent healthy subjects (human samples). e Western blotting analysis showing Cdon downregulation in muscle tissue from hindlimbs of Ash1L GT embryos at E18.5 compared to wild type (WT), and in C2C12 cells transfected with Ash1L siRNAs ( Ash1L KD) compared to control (NS). Densitometric analysis of both Ash1L and Cdon signals relative to vinculin as housekeeping protein are reported on the right. Paired one-tailed t test. Confidence intervals 95%. n = 3 (muscle tissue), three independent experiments (C2C12 cells). f Ash1L specifically binds to Cdon genomic region. Genome browser representation of Ash1L ChIP-seq peaks (from three replicates), at Cdon genomic region. Relative positions of both positive and negative regions are shown at the bottom (left). Chromatin immunoprecipitation on C2C12 cells transfected with non-targeting (NS) or Ash1L siRNAs ( Ash1L KD), and fixed at day 1 of differentiation, using Ash1L antibody and IgG as a negative control. qPCR analysis on positive and negative regions according to ChIP-sequencing results (right). Unpaired two-tailed t test. Confidence interval 95%. n = 3. Source data are provided as a Source Data file. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

    Journal: Nature Communications

    Article Title: The Trithorax protein Ash1L promotes myoblast fusion by activating Cdon expression

    doi: 10.1038/s41467-018-07313-8

    Figure Lengend Snippet: The fusion gene Cdon is a direct target of Ash1L. Cdon expression positively correlates with Ash1L during muscle development ( a ) and regeneration ( b ). RT-qPCR analysis on muscle tissue from hindlimbs of mice from the embryonic stage E16.5 to adulthood and in tibialis anterior of wild-type adult mice, untreated (UNT), or 5 and 10 days after cardiotoxin (CTX) injection. Unpaired two-tailed t test. Confidence intervals 95%. n = 6 ( a ), n = 4 ( b ). c Cdon protein level during in vitro muscle differentiation and in proliferating myoblasts vs. confluent cells. Immunoblot of C2C12 cells at days 0 and 1, and densitometric analysis of Cdon signal relative to vinculin as housekeeping protein (left panel). Comparison between proliferating myoblasts (P) and confluent cells (C) (right panel). Paired two-tailed t test. Confidence intervals 95%. Data are the mean for three independent experiments. d RT-qPCR analysis comparing Cdon expression in the presence and absence of Ash1L in three different models: muscle tissue from hindlimbs of wild-type (WT) and Ash1L GT embryos at E18.5, C2C12 cells transfected with non-targeting (NS) or Ash1L siRNAs ( Ash1L KD), and human primary cells transfected with non-targeting or Ash1L siRNAs, and collected at day 1 of differentiation. Unpaired two-tailed t test. Confidence intervals 95%. n = 6 (muscle tissue), four independent experiments (C2C12 cells), and three independent healthy subjects (human samples). e Western blotting analysis showing Cdon downregulation in muscle tissue from hindlimbs of Ash1L GT embryos at E18.5 compared to wild type (WT), and in C2C12 cells transfected with Ash1L siRNAs ( Ash1L KD) compared to control (NS). Densitometric analysis of both Ash1L and Cdon signals relative to vinculin as housekeeping protein are reported on the right. Paired one-tailed t test. Confidence intervals 95%. n = 3 (muscle tissue), three independent experiments (C2C12 cells). f Ash1L specifically binds to Cdon genomic region. Genome browser representation of Ash1L ChIP-seq peaks (from three replicates), at Cdon genomic region. Relative positions of both positive and negative regions are shown at the bottom (left). Chromatin immunoprecipitation on C2C12 cells transfected with non-targeting (NS) or Ash1L siRNAs ( Ash1L KD), and fixed at day 1 of differentiation, using Ash1L antibody and IgG as a negative control. qPCR analysis on positive and negative regions according to ChIP-sequencing results (right). Unpaired two-tailed t test. Confidence interval 95%. n = 3. Source data are provided as a Source Data file. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

    Article Snippet: The day after, cells were transfected with small interfering RNAs (siRNAs) against Ash1L or non-targeting control siRNAs [SMARTpool: ON-TARGETplus ASH1L siRNA L-020460 (human), L-041891 (mouse) or Non-Targeting Pool D-001810; Dharmacon], using Lipofectamine 3000 reagent (Thermo Fisher Scientific) following the manufacturer’s instructions.

    Techniques: Expressing, Quantitative RT-PCR, Mouse Assay, Injection, Two Tailed Test, In Vitro, Transfection, Western Blot, One-tailed Test, Chromatin Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction, Sequencing

    Effects of Ash1L ablation on myoblast fusion. a Primary myoblasts isolated from Ash1L GT mice display a fusion defect. Immunofluorescence for myosin heavy chain (in green) and nuclear staining (Hoechst) in primary cultures from wild-type (WT) and Ash1L GT mice at E18.5 stage, after inducing differentiation for 48 h. Scale bar, 100 µm. RT-qPCR analysis of Ash1L expression, validating Ash1L ablation in GT mice compared to wild-type mice. Percentage of Mhc-positive cells, calculated in comparison with the total number of nuclei. Fusion index, calculated as the number of nuclei present in myotubes (Mhc-positive and containing at least three nuclei) in comparison with the total number of nuclei. Nuclei distribution, calculated as the frequency of Mhc-positive cells containing the indicated number of nuclei. Unpaired two-tailed t test. Confidence intervals 95%. Results come from five biological replicates. b Ash1L knockdown leads to a muscle fusion defect. Immunofluorescence for myosin heavy chain and nuclear staining in C2C12 cells transfected with non-targeting (NS) and Ash1L siRNAs ( Ash1L KD) and differentiated for 3 days. Scale bar, 100 µm. Ash1L expression, percentage of Mhc-positive cells, fusion index, and nuclei distribution evaluated as described above. Unpaired two-tailed t test. Confidence intervals 95%. Results come from three biological replicates. c Muscle fusion defect upon Ash1L knockdown is conserved in human. Immunofluorescence for myosin heavy chain and nuclear staining in human primary myoblast transfected with non-targeting (human NS) and Ash1L siRNAs (human Ash1L KD) and differentiated for 4 days. Scale bar, 100 µm. Ash1L expression, percentage of MHC-positive cells, fusion index, and nuclei distribution evaluated as described above. Unpaired two-tailed t test. Confidence intervals 95%. Results come from three healthy subjects. Source data are provided as a Source Data file. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. NS not significant

    Journal: Nature Communications

    Article Title: The Trithorax protein Ash1L promotes myoblast fusion by activating Cdon expression

    doi: 10.1038/s41467-018-07313-8

    Figure Lengend Snippet: Effects of Ash1L ablation on myoblast fusion. a Primary myoblasts isolated from Ash1L GT mice display a fusion defect. Immunofluorescence for myosin heavy chain (in green) and nuclear staining (Hoechst) in primary cultures from wild-type (WT) and Ash1L GT mice at E18.5 stage, after inducing differentiation for 48 h. Scale bar, 100 µm. RT-qPCR analysis of Ash1L expression, validating Ash1L ablation in GT mice compared to wild-type mice. Percentage of Mhc-positive cells, calculated in comparison with the total number of nuclei. Fusion index, calculated as the number of nuclei present in myotubes (Mhc-positive and containing at least three nuclei) in comparison with the total number of nuclei. Nuclei distribution, calculated as the frequency of Mhc-positive cells containing the indicated number of nuclei. Unpaired two-tailed t test. Confidence intervals 95%. Results come from five biological replicates. b Ash1L knockdown leads to a muscle fusion defect. Immunofluorescence for myosin heavy chain and nuclear staining in C2C12 cells transfected with non-targeting (NS) and Ash1L siRNAs ( Ash1L KD) and differentiated for 3 days. Scale bar, 100 µm. Ash1L expression, percentage of Mhc-positive cells, fusion index, and nuclei distribution evaluated as described above. Unpaired two-tailed t test. Confidence intervals 95%. Results come from three biological replicates. c Muscle fusion defect upon Ash1L knockdown is conserved in human. Immunofluorescence for myosin heavy chain and nuclear staining in human primary myoblast transfected with non-targeting (human NS) and Ash1L siRNAs (human Ash1L KD) and differentiated for 4 days. Scale bar, 100 µm. Ash1L expression, percentage of MHC-positive cells, fusion index, and nuclei distribution evaluated as described above. Unpaired two-tailed t test. Confidence intervals 95%. Results come from three healthy subjects. Source data are provided as a Source Data file. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. NS not significant

    Article Snippet: The day after, cells were transfected with small interfering RNAs (siRNAs) against Ash1L or non-targeting control siRNAs [SMARTpool: ON-TARGETplus ASH1L siRNA L-020460 (human), L-041891 (mouse) or Non-Targeting Pool D-001810; Dharmacon], using Lipofectamine 3000 reagent (Thermo Fisher Scientific) following the manufacturer’s instructions.

    Techniques: Isolation, Mouse Assay, Immunofluorescence, Staining, Quantitative RT-PCR, Expressing, Two Tailed Test, Transfection