anti-h3k4me3 Millipore Search Results


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  • 99
    Millipore anti h3k4me3 millipore
    KDM5A knockdown enhanced osteogenic differentiation of MSCs. ( a ) qRT-PCR analysis and ( b ) western blot analysis of Kdm5a in MSCs after infected with lentiviral-Scrsh, lentiviral-Kdm5a-sh1 and lentiviral-Kdm5a-sh2. ( c ) Representative images of ALP staining of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( d ) Quantitative analysis of ALP activity of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( e ) Representative images of Alizarin red staining (including quantitative analysis) of MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 14 days of osteogenic induction. ( f ) qRT-PCR analysis and ( g ) western blot analysis of Col1a1, Ocn and Runx2 expression in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. ( h ) Immunostaining of Runx2 (red) location in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. Scale bar, 20 μ m. ( i ) Representative images of Alizarin red staining of MSCs isolated from OVX mice in Scrsh, Kdm5a-sh1 groups after 14 days of osteogenic induction. ( j ) Western blot analysis of <t>H3K4me3</t> expression in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor (JIB-04 with 300 nM) treatment. ( k ) qRT-PCR analysis of the expression of Col1a1, Ocn and Runx2 in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor treatment. ( l ) Representative images of Alizarin red staining of MSCs isolated from sham mice and OVX mice with or without Kdm5a inhibitor treatment. All the data were confirmed by three repeated tests. Data were mean±S.D. * P
    Anti H3k4me3 Millipore, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 337 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore h3k4me3 millipore
    KDM5A knockdown enhanced osteogenic differentiation of MSCs. ( a ) qRT-PCR analysis and ( b ) western blot analysis of Kdm5a in MSCs after infected with lentiviral-Scrsh, lentiviral-Kdm5a-sh1 and lentiviral-Kdm5a-sh2. ( c ) Representative images of ALP staining of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( d ) Quantitative analysis of ALP activity of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( e ) Representative images of Alizarin red staining (including quantitative analysis) of MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 14 days of osteogenic induction. ( f ) qRT-PCR analysis and ( g ) western blot analysis of Col1a1, Ocn and Runx2 expression in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. ( h ) Immunostaining of Runx2 (red) location in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. Scale bar, 20 μ m. ( i ) Representative images of Alizarin red staining of MSCs isolated from OVX mice in Scrsh, Kdm5a-sh1 groups after 14 days of osteogenic induction. ( j ) Western blot analysis of <t>H3K4me3</t> expression in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor (JIB-04 with 300 nM) treatment. ( k ) qRT-PCR analysis of the expression of Col1a1, Ocn and Runx2 in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor treatment. ( l ) Representative images of Alizarin red staining of MSCs isolated from sham mice and OVX mice with or without Kdm5a inhibitor treatment. All the data were confirmed by three repeated tests. Data were mean±S.D. * P
    H3k4me3 Millipore, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore anti h3k4me3 antibody upstate millipore
    KDM5A knockdown enhanced osteogenic differentiation of MSCs. ( a ) qRT-PCR analysis and ( b ) western blot analysis of Kdm5a in MSCs after infected with lentiviral-Scrsh, lentiviral-Kdm5a-sh1 and lentiviral-Kdm5a-sh2. ( c ) Representative images of ALP staining of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( d ) Quantitative analysis of ALP activity of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( e ) Representative images of Alizarin red staining (including quantitative analysis) of MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 14 days of osteogenic induction. ( f ) qRT-PCR analysis and ( g ) western blot analysis of Col1a1, Ocn and Runx2 expression in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. ( h ) Immunostaining of Runx2 (red) location in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. Scale bar, 20 μ m. ( i ) Representative images of Alizarin red staining of MSCs isolated from OVX mice in Scrsh, Kdm5a-sh1 groups after 14 days of osteogenic induction. ( j ) Western blot analysis of <t>H3K4me3</t> expression in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor (JIB-04 with 300 nM) treatment. ( k ) qRT-PCR analysis of the expression of Col1a1, Ocn and Runx2 in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor treatment. ( l ) Representative images of Alizarin red staining of MSCs isolated from sham mice and OVX mice with or without Kdm5a inhibitor treatment. All the data were confirmed by three repeated tests. Data were mean±S.D. * P
    Anti H3k4me3 Antibody Upstate Millipore, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore h3k4me3 millipore 04745 antibody
    KDM5A knockdown enhanced osteogenic differentiation of MSCs. ( a ) qRT-PCR analysis and ( b ) western blot analysis of Kdm5a in MSCs after infected with lentiviral-Scrsh, lentiviral-Kdm5a-sh1 and lentiviral-Kdm5a-sh2. ( c ) Representative images of ALP staining of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( d ) Quantitative analysis of ALP activity of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( e ) Representative images of Alizarin red staining (including quantitative analysis) of MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 14 days of osteogenic induction. ( f ) qRT-PCR analysis and ( g ) western blot analysis of Col1a1, Ocn and Runx2 expression in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. ( h ) Immunostaining of Runx2 (red) location in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. Scale bar, 20 μ m. ( i ) Representative images of Alizarin red staining of MSCs isolated from OVX mice in Scrsh, Kdm5a-sh1 groups after 14 days of osteogenic induction. ( j ) Western blot analysis of <t>H3K4me3</t> expression in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor (JIB-04 with 300 nM) treatment. ( k ) qRT-PCR analysis of the expression of Col1a1, Ocn and Runx2 in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor treatment. ( l ) Representative images of Alizarin red staining of MSCs isolated from sham mice and OVX mice with or without Kdm5a inhibitor treatment. All the data were confirmed by three repeated tests. Data were mean±S.D. * P
    H3k4me3 Millipore 04745 Antibody, supplied by Millipore, 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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    92
    Merck KGaA anti h3k4me3
    Relative enrichment of <t>H3K4me3</t> and H3K27me3 for E. grandis homologs of transcription factors and signal peptides regulating vascular development and flowering in developing secondary xylem tissue. Blue bars, enrichment of H3K4 and H3K27 trimethylation (shown as N-terminal region of histone H3), represented as a signal of zero (no significant enrichment) to four bars (16-fold enrichment). Red bars, absolute RNA-seq expression levels (max FPKM value, 228) for roots, phloem, developing secondary xylem (DSX), shoots, young leaves (YL), mature leaves (ML) and flowers 41 , 42 .
    Anti H3k4me3, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Active Motif h3k4me3
    Lack of Ccl1 results in decreased global <t>H3K4me3.</t> The Fusarium wild type strains ( F. fujikuroi – FfWT, F. graminearum – FgWT) and the respective CCL1 deletion mutants ( F. fujikuroi – Δ ffccl1 , F. graminearum – Δ fgccl1 ) were grown for 3 days on solid complete medium. Whole protein extracts were subsequently isolated from lyophilized mycelia and roughly 15 μg of proteins were used for SDS-Page and western blotting. (A) The following primary antibodies were used for detection: H3 C-Term, H3K4me1, H3K4me2 as well as three different H3K4me3. (B) Coomassie staining was performed as an additional loading control.
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    Millipore chip qualified antibodies
    Lack of Ccl1 results in decreased global <t>H3K4me3.</t> The Fusarium wild type strains ( F. fujikuroi – FfWT, F. graminearum – FgWT) and the respective CCL1 deletion mutants ( F. fujikuroi – Δ ffccl1 , F. graminearum – Δ fgccl1 ) were grown for 3 days on solid complete medium. Whole protein extracts were subsequently isolated from lyophilized mycelia and roughly 15 μg of proteins were used for SDS-Page and western blotting. (A) The following primary antibodies were used for detection: H3 C-Term, H3K4me1, H3K4me2 as well as three different H3K4me3. (B) Coomassie staining was performed as an additional loading control.
    Chip Qualified Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti h3k4me3 no 17 614 antibody
    Lack of Ccl1 results in decreased global <t>H3K4me3.</t> The Fusarium wild type strains ( F. fujikuroi – FfWT, F. graminearum – FgWT) and the respective CCL1 deletion mutants ( F. fujikuroi – Δ ffccl1 , F. graminearum – Δ fgccl1 ) were grown for 3 days on solid complete medium. Whole protein extracts were subsequently isolated from lyophilized mycelia and roughly 15 μg of proteins were used for SDS-Page and western blotting. (A) The following primary antibodies were used for detection: H3 C-Term, H3K4me1, H3K4me2 as well as three different H3K4me3. (B) Coomassie staining was performed as an additional loading control.
    Anti H3k4me3 No 17 614 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse anti h3k4me3 mc315
    Lack of Ccl1 results in decreased global <t>H3K4me3.</t> The Fusarium wild type strains ( F. fujikuroi – FfWT, F. graminearum – FgWT) and the respective CCL1 deletion mutants ( F. fujikuroi – Δ ffccl1 , F. graminearum – Δ fgccl1 ) were grown for 3 days on solid complete medium. Whole protein extracts were subsequently isolated from lyophilized mycelia and roughly 15 μg of proteins were used for SDS-Page and western blotting. (A) The following primary antibodies were used for detection: H3 C-Term, H3K4me1, H3K4me2 as well as three different H3K4me3. (B) Coomassie staining was performed as an additional loading control.
    Mouse Anti H3k4me3 Mc315, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse anti h3k4me3
    Lack of Ccl1 results in decreased global <t>H3K4me3.</t> The Fusarium wild type strains ( F. fujikuroi – FfWT, F. graminearum – FgWT) and the respective CCL1 deletion mutants ( F. fujikuroi – Δ ffccl1 , F. graminearum – Δ fgccl1 ) were grown for 3 days on solid complete medium. Whole protein extracts were subsequently isolated from lyophilized mycelia and roughly 15 μg of proteins were used for SDS-Page and western blotting. (A) The following primary antibodies were used for detection: H3 C-Term, H3K4me1, H3K4me2 as well as three different H3K4me3. (B) Coomassie staining was performed as an additional loading control.
    Mouse Anti H3k4me3, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam h3k4me3
    Reduction of <t>H3K4me3</t> does not affect expression of regulated genes a , Distribution of ASH2 binding in stable (red), regulated (blue) and silent genes (grey). b , H3K4me3 is strongly decreased in ash2 I1 mutant clones in WID. c , En immunostaining in WID (merged). The scale bar represents 20 μm. d , The levels of the stable gene En are reduced in mutant clones. e, GFP negative cells indicate ash2 I1 mutant cells in c and d . f , CycA immunostaining in WID (merged). The scale bar represents 20 μm. g, CycA is decreased in ash2 I1 mutant clones. h, GFP negative cells indicate ash2 I1 mutant cells in f and g . i, pdm2 fluorescence in situ hybridization in ash2 I1 mutant clones in WID (merged). The scale bar represents 20 μm. j , No changes in pdm2 expression are observed in ash2 I1 mutant clones. k, GFP negative cells indicate ash2 I1 mutant cells in i and j . l, Boss immunostaining in EID. The scale bar represents 20 μm. m, Optical cross-section (white line in l ) showing Boss in all R8 photoreceptor cells (merged). n , No changes in Boss expression are observed in ash2 I1 mutant clones. o, GFP negative cells indicate ash2 I1 mutant cells in m and n . p , Diagram summarizing the result in m – o . Green cells express the wild-type ash2 allele and black cells correspond to homozygous ash2 I1 mutant cells. Boss (magenta cap) localizes in the apical side of R8.
    H3k4me3, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 3388 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore mouse antibody
    Reduction of <t>H3K4me3</t> does not affect expression of regulated genes a , Distribution of ASH2 binding in stable (red), regulated (blue) and silent genes (grey). b , H3K4me3 is strongly decreased in ash2 I1 mutant clones in WID. c , En immunostaining in WID (merged). The scale bar represents 20 μm. d , The levels of the stable gene En are reduced in mutant clones. e, GFP negative cells indicate ash2 I1 mutant cells in c and d . f , CycA immunostaining in WID (merged). The scale bar represents 20 μm. g, CycA is decreased in ash2 I1 mutant clones. h, GFP negative cells indicate ash2 I1 mutant cells in f and g . i, pdm2 fluorescence in situ hybridization in ash2 I1 mutant clones in WID (merged). The scale bar represents 20 μm. j , No changes in pdm2 expression are observed in ash2 I1 mutant clones. k, GFP negative cells indicate ash2 I1 mutant cells in i and j . l, Boss immunostaining in EID. The scale bar represents 20 μm. m, Optical cross-section (white line in l ) showing Boss in all R8 photoreceptor cells (merged). n , No changes in Boss expression are observed in ash2 I1 mutant clones. o, GFP negative cells indicate ash2 I1 mutant cells in m and n . p , Diagram summarizing the result in m – o . Green cells express the wild-type ash2 allele and black cells correspond to homozygous ash2 I1 mutant cells. Boss (magenta cap) localizes in the apical side of R8.
    Mouse Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1551 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore h3k4
    Reduction of <t>H3K4me3</t> does not affect expression of regulated genes a , Distribution of ASH2 binding in stable (red), regulated (blue) and silent genes (grey). b , H3K4me3 is strongly decreased in ash2 I1 mutant clones in WID. c , En immunostaining in WID (merged). The scale bar represents 20 μm. d , The levels of the stable gene En are reduced in mutant clones. e, GFP negative cells indicate ash2 I1 mutant cells in c and d . f , CycA immunostaining in WID (merged). The scale bar represents 20 μm. g, CycA is decreased in ash2 I1 mutant clones. h, GFP negative cells indicate ash2 I1 mutant cells in f and g . i, pdm2 fluorescence in situ hybridization in ash2 I1 mutant clones in WID (merged). The scale bar represents 20 μm. j , No changes in pdm2 expression are observed in ash2 I1 mutant clones. k, GFP negative cells indicate ash2 I1 mutant cells in i and j . l, Boss immunostaining in EID. The scale bar represents 20 μm. m, Optical cross-section (white line in l ) showing Boss in all R8 photoreceptor cells (merged). n , No changes in Boss expression are observed in ash2 I1 mutant clones. o, GFP negative cells indicate ash2 I1 mutant cells in m and n . p , Diagram summarizing the result in m – o . Green cells express the wild-type ash2 allele and black cells correspond to homozygous ash2 I1 mutant cells. Boss (magenta cap) localizes in the apical side of R8.
    H3k4, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 141 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    KDM5A knockdown enhanced osteogenic differentiation of MSCs. ( a ) qRT-PCR analysis and ( b ) western blot analysis of Kdm5a in MSCs after infected with lentiviral-Scrsh, lentiviral-Kdm5a-sh1 and lentiviral-Kdm5a-sh2. ( c ) Representative images of ALP staining of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( d ) Quantitative analysis of ALP activity of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( e ) Representative images of Alizarin red staining (including quantitative analysis) of MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 14 days of osteogenic induction. ( f ) qRT-PCR analysis and ( g ) western blot analysis of Col1a1, Ocn and Runx2 expression in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. ( h ) Immunostaining of Runx2 (red) location in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. Scale bar, 20 μ m. ( i ) Representative images of Alizarin red staining of MSCs isolated from OVX mice in Scrsh, Kdm5a-sh1 groups after 14 days of osteogenic induction. ( j ) Western blot analysis of H3K4me3 expression in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor (JIB-04 with 300 nM) treatment. ( k ) qRT-PCR analysis of the expression of Col1a1, Ocn and Runx2 in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor treatment. ( l ) Representative images of Alizarin red staining of MSCs isolated from sham mice and OVX mice with or without Kdm5a inhibitor treatment. All the data were confirmed by three repeated tests. Data were mean±S.D. * P

    Journal: Cell Death & Disease

    Article Title: KDM5A controls bone morphogenic protein 2-induced osteogenic differentiation of bone mesenchymal stem cells during osteoporosis

    doi: 10.1038/cddis.2016.238

    Figure Lengend Snippet: KDM5A knockdown enhanced osteogenic differentiation of MSCs. ( a ) qRT-PCR analysis and ( b ) western blot analysis of Kdm5a in MSCs after infected with lentiviral-Scrsh, lentiviral-Kdm5a-sh1 and lentiviral-Kdm5a-sh2. ( c ) Representative images of ALP staining of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( d ) Quantitative analysis of ALP activity of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( e ) Representative images of Alizarin red staining (including quantitative analysis) of MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 14 days of osteogenic induction. ( f ) qRT-PCR analysis and ( g ) western blot analysis of Col1a1, Ocn and Runx2 expression in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. ( h ) Immunostaining of Runx2 (red) location in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. Scale bar, 20 μ m. ( i ) Representative images of Alizarin red staining of MSCs isolated from OVX mice in Scrsh, Kdm5a-sh1 groups after 14 days of osteogenic induction. ( j ) Western blot analysis of H3K4me3 expression in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor (JIB-04 with 300 nM) treatment. ( k ) qRT-PCR analysis of the expression of Col1a1, Ocn and Runx2 in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor treatment. ( l ) Representative images of Alizarin red staining of MSCs isolated from sham mice and OVX mice with or without Kdm5a inhibitor treatment. All the data were confirmed by three repeated tests. Data were mean±S.D. * P

    Article Snippet: Immunoprecipitation (IP) was carried out overnight with purified anti-H3K4me3 and SMAD5 antibody (Millipore, Bedford, MA, USA) or normal mouse IgG as a negative control.

    Techniques: Quantitative RT-PCR, Western Blot, Infection, ALP Assay, Staining, Activity Assay, Expressing, Immunostaining, Isolation, Mouse Assay

    KDM5A inhibited Runx2 expression in MSC by removal of H3K4me3 marks. ( a ) Western blot analysis of p-Smad1/5/8, Smad1 and Smad4 in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 4 hours of osteogenic induction. ( b ) Quantitative analysis of p-Smad1 expression. Smad1 was used as internal control. ( c ) Immunostaining of p-Smad1/5/8 (green) location in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 4 hours of osteogenic induction. Scale bar, 20 μ m. ( d ) Schematics of Runx2 promoter denoting ChIP-PCR amplified region (−1105 bp to −1065 bp) encompassing the SMAD binding element and the control region 6-kb upstream of the transcription start site (−6173 bp to −6034 bp). ( e ) Western blot analysis of H3K4me3 in MSCs after 0, 3, 7 and 14 days BMP2 treatment. ( f ) Occupancy of H3K4me3 at the Runx2 promoter following BMP2 treatment. ( g ) SMAD5 occupancy at the Runx2 promoter after BMP2 treatment. ( h ) Western blot analysis of H3K4me3 in MSCs of sham and OVX mice. ( i ) Occupancy of H3K4me3 at the Runx2 promoter in MSCs of sham and OVX mice following BMP2 treatment. ( j ) Knockdown of Kdm5a increased the occupancy of H3K4me3 at the Runx2 promoter following BMP2 treatment in MSCs of sham and OVX mice. ( k ) Overexpression Kdm5a decreased the occupancy of H3K4me3 at the Runx2 promoter following BMP2 treatment in MSCs of sham and OVX mice. All the data were confirmed by three repeated tests. Data were mean±S.D. ** P

    Journal: Cell Death & Disease

    Article Title: KDM5A controls bone morphogenic protein 2-induced osteogenic differentiation of bone mesenchymal stem cells during osteoporosis

    doi: 10.1038/cddis.2016.238

    Figure Lengend Snippet: KDM5A inhibited Runx2 expression in MSC by removal of H3K4me3 marks. ( a ) Western blot analysis of p-Smad1/5/8, Smad1 and Smad4 in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 4 hours of osteogenic induction. ( b ) Quantitative analysis of p-Smad1 expression. Smad1 was used as internal control. ( c ) Immunostaining of p-Smad1/5/8 (green) location in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 4 hours of osteogenic induction. Scale bar, 20 μ m. ( d ) Schematics of Runx2 promoter denoting ChIP-PCR amplified region (−1105 bp to −1065 bp) encompassing the SMAD binding element and the control region 6-kb upstream of the transcription start site (−6173 bp to −6034 bp). ( e ) Western blot analysis of H3K4me3 in MSCs after 0, 3, 7 and 14 days BMP2 treatment. ( f ) Occupancy of H3K4me3 at the Runx2 promoter following BMP2 treatment. ( g ) SMAD5 occupancy at the Runx2 promoter after BMP2 treatment. ( h ) Western blot analysis of H3K4me3 in MSCs of sham and OVX mice. ( i ) Occupancy of H3K4me3 at the Runx2 promoter in MSCs of sham and OVX mice following BMP2 treatment. ( j ) Knockdown of Kdm5a increased the occupancy of H3K4me3 at the Runx2 promoter following BMP2 treatment in MSCs of sham and OVX mice. ( k ) Overexpression Kdm5a decreased the occupancy of H3K4me3 at the Runx2 promoter following BMP2 treatment in MSCs of sham and OVX mice. All the data were confirmed by three repeated tests. Data were mean±S.D. ** P

    Article Snippet: Immunoprecipitation (IP) was carried out overnight with purified anti-H3K4me3 and SMAD5 antibody (Millipore, Bedford, MA, USA) or normal mouse IgG as a negative control.

    Techniques: Expressing, Western Blot, Infection, Plasmid Preparation, Immunostaining, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Binding Assay, Mouse Assay, Over Expression

    KDM5A overexpression impaired osteogenic differentiation of MSCs. ( a ) qRT-PCR analysis and ( b ) western blot analysis of Kdm5a in MSCs after 0, 3, 7 and 14 days osteogenic induction. ( c ) qRT-PCR analysis and ( d ) western blot analysis of Kdm5a in MSCs after infected with lentiviral vector (MSC/V) and lentiviral-Kdm5a (MSC/KDM5A). ( e ) Western blot analysis of H3K4me3, H3K9me3 and H3K27me3 in MSCs with overexpression of Kdm5a. ( f ) ALP activity o f MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 7 days of osteogenic induction were detected with ALP staining and quantified. ( g ) Mineralized nodules formed by MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 14 days of osteogenic induction were detected with Alizarin red staining and quantified. ( h ) qRT-PCR analysis and ( i ) western blot analysis of Col1a1, Ocn and Runx2 expression in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 7 days of osteogenic induction. ( j ) Immunostaining of RUNX2 (red) location in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 7 days of osteogenic induction. Scale bar, 20 μ m. All the data were confirmed by three repeated tests. Data were mean±S.D. ** P

    Journal: Cell Death & Disease

    Article Title: KDM5A controls bone morphogenic protein 2-induced osteogenic differentiation of bone mesenchymal stem cells during osteoporosis

    doi: 10.1038/cddis.2016.238

    Figure Lengend Snippet: KDM5A overexpression impaired osteogenic differentiation of MSCs. ( a ) qRT-PCR analysis and ( b ) western blot analysis of Kdm5a in MSCs after 0, 3, 7 and 14 days osteogenic induction. ( c ) qRT-PCR analysis and ( d ) western blot analysis of Kdm5a in MSCs after infected with lentiviral vector (MSC/V) and lentiviral-Kdm5a (MSC/KDM5A). ( e ) Western blot analysis of H3K4me3, H3K9me3 and H3K27me3 in MSCs with overexpression of Kdm5a. ( f ) ALP activity o f MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 7 days of osteogenic induction were detected with ALP staining and quantified. ( g ) Mineralized nodules formed by MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 14 days of osteogenic induction were detected with Alizarin red staining and quantified. ( h ) qRT-PCR analysis and ( i ) western blot analysis of Col1a1, Ocn and Runx2 expression in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 7 days of osteogenic induction. ( j ) Immunostaining of RUNX2 (red) location in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 7 days of osteogenic induction. Scale bar, 20 μ m. All the data were confirmed by three repeated tests. Data were mean±S.D. ** P

    Article Snippet: Immunoprecipitation (IP) was carried out overnight with purified anti-H3K4me3 and SMAD5 antibody (Millipore, Bedford, MA, USA) or normal mouse IgG as a negative control.

    Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Infection, Plasmid Preparation, ALP Assay, Activity Assay, Staining, Expressing, Immunostaining

    Cystic EBs lack epigenetic features of visceral yolk sac endoderm. (A) Global DNA methylation in E12.5 yolk sac endoderm (ysE) is lower than in d15 cystic EBs, which shows greater similarity with the DNA methylation level of E12.5 fetal liver. Whole genome bisulfite sequencing (WGBS) data was analyzed using 5 kb windows across the genome. Box plots show the range of DNA methylation for each tissue indicating the median and the interquartile range (IQR). (B) Cystic EBs (d15) cluster closer to E12.5 fetal liver than E12.5 ysE by DNA methylation levels. Hierarchical clustering was done using 5 kb windows ( n =4996) that showed the greatest similarity between replicates, but differed with at least one other tissue (details in Supplementary materials). (C) De novo DNA methyltransferases Dnmt3b and Dnmt3l are more highly expressed (reads per kilobase per million reads (RPKM)) in RNA-seq data from cystic EBs than in E9.5, 12.5 ysE and fetal liver. (D) IAPEz repeats are highly methylated, but show a lower level of DNA methylation in E12.5 ysE compared to d15 cystic EBs, which show levels more similar to E12.5 fetal liver. LINE repeats are less methylated, but show a similar pattern with E12.5 ysE showing a lower level of DNA methylation than d15 cystic EBs, which show levels more similar to fetal liver. Box plots (shaded as in 3A) show the percentage of methylation for each sample indicting the median and IQR. (E) E12.5 ysE expresses IAPez repeats at a higher level than cystic EBs, while SINEs, LINEs, and all repeats show no difference in expression. The Kernel density plot displays the probability of repeats showing different expression in these two tissues ( x -axis: log 2 fold difference in expression, negative values represent overexpression in E12.5 ysE, positive values represent overexpression in cystic EBs). (F) The expression of selected genes from chromatin modifying complexes from RNA-seq data. (G) No difference in the global level of histone modifications was detected by Western blot analysis for H3K4me3, H3K27ac, H3K27me3, H3K9me3 and H3K9me2 histone modifications in undifferentiated ES cells, cystic EBs d15, E12.5 ysE, E12.5 total VYS and E12.5 fetal liver. A pan-H3 antibody is provided below each of the modifications as a loading control. Error bars in (C) and (F) show the standard error of the mean (* P ≤0.05, ** P ≤0.01, and *** P ≤0.001).

    Journal: Developmental Biology

    Article Title: Imprinted expression in cystic embryoid bodies shows an embryonic and not an extra-embryonic pattern

    doi: 10.1016/j.ydbio.2015.04.010

    Figure Lengend Snippet: Cystic EBs lack epigenetic features of visceral yolk sac endoderm. (A) Global DNA methylation in E12.5 yolk sac endoderm (ysE) is lower than in d15 cystic EBs, which shows greater similarity with the DNA methylation level of E12.5 fetal liver. Whole genome bisulfite sequencing (WGBS) data was analyzed using 5 kb windows across the genome. Box plots show the range of DNA methylation for each tissue indicating the median and the interquartile range (IQR). (B) Cystic EBs (d15) cluster closer to E12.5 fetal liver than E12.5 ysE by DNA methylation levels. Hierarchical clustering was done using 5 kb windows ( n =4996) that showed the greatest similarity between replicates, but differed with at least one other tissue (details in Supplementary materials). (C) De novo DNA methyltransferases Dnmt3b and Dnmt3l are more highly expressed (reads per kilobase per million reads (RPKM)) in RNA-seq data from cystic EBs than in E9.5, 12.5 ysE and fetal liver. (D) IAPEz repeats are highly methylated, but show a lower level of DNA methylation in E12.5 ysE compared to d15 cystic EBs, which show levels more similar to E12.5 fetal liver. LINE repeats are less methylated, but show a similar pattern with E12.5 ysE showing a lower level of DNA methylation than d15 cystic EBs, which show levels more similar to fetal liver. Box plots (shaded as in 3A) show the percentage of methylation for each sample indicting the median and IQR. (E) E12.5 ysE expresses IAPez repeats at a higher level than cystic EBs, while SINEs, LINEs, and all repeats show no difference in expression. The Kernel density plot displays the probability of repeats showing different expression in these two tissues ( x -axis: log 2 fold difference in expression, negative values represent overexpression in E12.5 ysE, positive values represent overexpression in cystic EBs). (F) The expression of selected genes from chromatin modifying complexes from RNA-seq data. (G) No difference in the global level of histone modifications was detected by Western blot analysis for H3K4me3, H3K27ac, H3K27me3, H3K9me3 and H3K9me2 histone modifications in undifferentiated ES cells, cystic EBs d15, E12.5 ysE, E12.5 total VYS and E12.5 fetal liver. A pan-H3 antibody is provided below each of the modifications as a loading control. Error bars in (C) and (F) show the standard error of the mean (* P ≤0.05, ** P ≤0.01, and *** P ≤0.001).

    Article Snippet: Native ChIP for H3K4me3 (antibody: cat. 07-473, lot 0608038479, Millipore) was conducted by homogenizing 20× VYS under liquid nitrogen (E12.5 FVB/N pooled from 2 litters) and then proceeding as previously described ( ).

    Techniques: DNA Methylation Assay, Methylation Sequencing, RNA Sequencing Assay, Methylation, Expressing, Over Expression, Western Blot

    Status of histone modifications and pol II binding in the proximal regions of STAT6 target TSSs. (A) Distribution of the averaged ChIP Seq tags for H3K4me3 (top panels), H3Ac (middle panels) and pol II (bottom panels) in Ramos cells. Data from active target genes (STAT6 binding plus TSS induction in Ramos cells) are shown in the left panels, and data from silent target genes (STAT6 binding plus TSS induction in BEAS2B cells but both negative in Ramos cells) are shown in the right panels. Blue, green, red and purple lines indicate the results for the IP (IL-4 (+)), IP (IL-4 (−)), WCE (IL-4 (+)) and WCE (IL-4 (−)) experiments, respectively. On the x -axis, the position of the associated TSS is designated as zero. (B) Results of an analysis similar to that shown in (A) in BEAS2B cells.

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    Article Title: Characterization of STAT6 Target Genes in Human B Cells and Lung Epithelial Cells

    doi: 10.1093/dnares/dsr025

    Figure Lengend Snippet: Status of histone modifications and pol II binding in the proximal regions of STAT6 target TSSs. (A) Distribution of the averaged ChIP Seq tags for H3K4me3 (top panels), H3Ac (middle panels) and pol II (bottom panels) in Ramos cells. Data from active target genes (STAT6 binding plus TSS induction in Ramos cells) are shown in the left panels, and data from silent target genes (STAT6 binding plus TSS induction in BEAS2B cells but both negative in Ramos cells) are shown in the right panels. Blue, green, red and purple lines indicate the results for the IP (IL-4 (+)), IP (IL-4 (−)), WCE (IL-4 (+)) and WCE (IL-4 (−)) experiments, respectively. On the x -axis, the position of the associated TSS is designated as zero. (B) Results of an analysis similar to that shown in (A) in BEAS2B cells.

    Article Snippet: Anti-STAT-6 antibody (Santa Cruz, M-20), anti-RNA pol II antibody (Abcam, ab817), anti-H3K4me3 antibody (ab1012) and anti-H3Ac antibody (Millipore, 06–599) were used for the indicated experiments.

    Techniques: Binding Assay, Chromatin Immunoprecipitation

    DLL4-enriched permissive histone mark H3K4me3 around Foxp3 promoter and consensus non-coding sequences during iTreg differentiation. a 2 × 10 6 of naive CD4 T cells were skewed toward Th0 or iT reg differentiation with or without DLL4 stimulation in vitro. Chromatin immunoprecipitation were performed to detect H3K4me3 around Foxp3 promoter, consensus non-coding sequence (CNS)1, CNS2, CNS3 after 72 h. b Changes of H3K4me and H3K4me3 by DLL4 stimulation during iT reg differentiation was detected at 48 h of skewing. c H3K4me3 kinetics at Foxp3 CNS1 after 6 h, 24 h, 48 h and 72 h post iT reg differentiation were measured with or without DLL4 stimulation in vitro. Data represent mean ± SEM. Data were from one experiment representative of two to three experiments. * P

    Journal: Mucosal immunology

    Article Title: Notch ligand Delta-like 4 induces epigenetic regulation of Treg cell differentiation and function in viral infection

    doi: 10.1038/s41385-018-0052-1

    Figure Lengend Snippet: DLL4-enriched permissive histone mark H3K4me3 around Foxp3 promoter and consensus non-coding sequences during iTreg differentiation. a 2 × 10 6 of naive CD4 T cells were skewed toward Th0 or iT reg differentiation with or without DLL4 stimulation in vitro. Chromatin immunoprecipitation were performed to detect H3K4me3 around Foxp3 promoter, consensus non-coding sequence (CNS)1, CNS2, CNS3 after 72 h. b Changes of H3K4me and H3K4me3 by DLL4 stimulation during iT reg differentiation was detected at 48 h of skewing. c H3K4me3 kinetics at Foxp3 CNS1 after 6 h, 24 h, 48 h and 72 h post iT reg differentiation were measured with or without DLL4 stimulation in vitro. Data represent mean ± SEM. Data were from one experiment representative of two to three experiments. * P

    Article Snippet: After three washes, sample were labeled with primary antibody anti-H3K4me3 (Millipore #07-473) in 1:200 dilution for 30 min at room temperature and secondary antibody antigen presenting cell (APC) or fluorescein isothiocyanate-antirabbit antibody for 20 min at room temperature.

    Techniques: In Vitro, Chromatin Immunoprecipitation, Sequencing

    Smyd3 regulated DLL4-enhanced iT reg differentiation and function in vitro. a Naive CD4 T cells (10 6 ) from wild-type B6 spleen were activated (Th0) or differentiated to Th1, Th2, Th17, Th9, iT reg , and IL-27 T R 1. After 72 h, SMYD3 were blotted. b Naive CD4 T cells (10 6 ) from Cre− control or Cre + CD4-specific SMYD3 conditional knockout (cKO) were differentiated to iT reg . After 72 h, SMYD3 level were quantified by western blotting. c Smyd3 mRNA level were measure in iT reg and DLL4-stimulated iT reg differentiation at 24, 48, and 72 h from Cre− control and Cre + Smyd3 cKO. d Naive CD4 T cells (2 × 10 6 ) from Cre− control with or without DLL4 stimulation or Cre + CD4-specific SMYD3 were differentiated to iT reg . After 72 h, H3K4me3 were precipitated, and Foxp3 promoter and CNS1, 2, 3 were qPCR quantified compared with input control. e Naive CD4 T cells (10 6 ) from Cre− control or Cre + SMYD3 cKO were activated (Th0) or differentiated to iT reg with or without DLL4. After 72 h of differentiation, Foxp3 and CD25 were labeled and quantified by flow cytometry. f Naive CD4 T cells (10 6 ) from Cre− control or Cre + SMYD3 cKO were activated (Th0) or differentiated to iT reg with or without DLL4. After 72 h of differentiation, both Th0 and iT reg were rested in IL-2 10 ng/mL for another 72 h. Foxp3 were detected and quantified by flow cytometry. g After 6 days of iT reg differentiation with DLL4 as described in f , viable DLL4-exposed iT reg were sorted out as DAPI − CD25+ and co-cultured with Cell Trace violet (CTV) labeled CD45.1+ naive T cells with anti-CD3/anti-CD28 beads. After 3 days in co-culture, proliferation was assessed by CTV dilution in CD45.1 + responder cells. Data represent mean ± SEM. Data were from one experiment representative of two to three experiments. * P

    Journal: Mucosal immunology

    Article Title: Notch ligand Delta-like 4 induces epigenetic regulation of Treg cell differentiation and function in viral infection

    doi: 10.1038/s41385-018-0052-1

    Figure Lengend Snippet: Smyd3 regulated DLL4-enhanced iT reg differentiation and function in vitro. a Naive CD4 T cells (10 6 ) from wild-type B6 spleen were activated (Th0) or differentiated to Th1, Th2, Th17, Th9, iT reg , and IL-27 T R 1. After 72 h, SMYD3 were blotted. b Naive CD4 T cells (10 6 ) from Cre− control or Cre + CD4-specific SMYD3 conditional knockout (cKO) were differentiated to iT reg . After 72 h, SMYD3 level were quantified by western blotting. c Smyd3 mRNA level were measure in iT reg and DLL4-stimulated iT reg differentiation at 24, 48, and 72 h from Cre− control and Cre + Smyd3 cKO. d Naive CD4 T cells (2 × 10 6 ) from Cre− control with or without DLL4 stimulation or Cre + CD4-specific SMYD3 were differentiated to iT reg . After 72 h, H3K4me3 were precipitated, and Foxp3 promoter and CNS1, 2, 3 were qPCR quantified compared with input control. e Naive CD4 T cells (10 6 ) from Cre− control or Cre + SMYD3 cKO were activated (Th0) or differentiated to iT reg with or without DLL4. After 72 h of differentiation, Foxp3 and CD25 were labeled and quantified by flow cytometry. f Naive CD4 T cells (10 6 ) from Cre− control or Cre + SMYD3 cKO were activated (Th0) or differentiated to iT reg with or without DLL4. After 72 h of differentiation, both Th0 and iT reg were rested in IL-2 10 ng/mL for another 72 h. Foxp3 were detected and quantified by flow cytometry. g After 6 days of iT reg differentiation with DLL4 as described in f , viable DLL4-exposed iT reg were sorted out as DAPI − CD25+ and co-cultured with Cell Trace violet (CTV) labeled CD45.1+ naive T cells with anti-CD3/anti-CD28 beads. After 3 days in co-culture, proliferation was assessed by CTV dilution in CD45.1 + responder cells. Data represent mean ± SEM. Data were from one experiment representative of two to three experiments. * P

    Article Snippet: After three washes, sample were labeled with primary antibody anti-H3K4me3 (Millipore #07-473) in 1:200 dilution for 30 min at room temperature and secondary antibody antigen presenting cell (APC) or fluorescein isothiocyanate-antirabbit antibody for 20 min at room temperature.

    Techniques: In Vitro, Knock-Out, Western Blot, Real-time Polymerase Chain Reaction, Labeling, Flow Cytometry, Cytometry, Cell Culture, Co-Culture Assay

    Effect of IL-13 stimulation and STAT6 silencing on epigenetic status of NTRK1 In A , levels of H3K9Ac, H3K27Ac, and H3K4me3 in the promoters of CCL26 , NTRK1 , and MYOD after IL-13 stimulation were quantified by ChIP-RT-PCR. Data from 3 independent experiments calculated as percentage of signal in input DNA normalized to the level of signal in PPIA gene are presented as mean values with standard error measurements. In B , levels of histone modification in the NTRK1 promoter following induction with IL-13 for 24 hr were quantified by RT-PCR in control (Ctrl) and TE-7 cells where the STAT6 gene was silenced by shRNA (STAT6KD). Combined data for 2 independent experiments are shown. *p

    Journal: Mucosal immunology

    Article Title: Neurotrophic tyrosine kinase receptor 1 is a direct transcriptional and epigenetic target of IL-13 involved in allergic inflammation

    doi: 10.1038/mi.2014.109

    Figure Lengend Snippet: Effect of IL-13 stimulation and STAT6 silencing on epigenetic status of NTRK1 In A , levels of H3K9Ac, H3K27Ac, and H3K4me3 in the promoters of CCL26 , NTRK1 , and MYOD after IL-13 stimulation were quantified by ChIP-RT-PCR. Data from 3 independent experiments calculated as percentage of signal in input DNA normalized to the level of signal in PPIA gene are presented as mean values with standard error measurements. In B , levels of histone modification in the NTRK1 promoter following induction with IL-13 for 24 hr were quantified by RT-PCR in control (Ctrl) and TE-7 cells where the STAT6 gene was silenced by shRNA (STAT6KD). Combined data for 2 independent experiments are shown. *p

    Article Snippet: Some ChIP experiments were performed with H3K27Ac antibody from Diagenode (pAb-196-050, Diagenode Inc., Denville, NJ) and H3K4me3 antibody from Millipore (17–614, Billerica, MA).

    Techniques: Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Modification, shRNA

    Transcriptional deregulation and mislocalization of inactive histone marks is induced in cells exposed to softer matrices. ( A ) Experimental scheme. RNA sequencing was performed in two independent biological replicates (Pearson correlation coefficient: 2 kPa replicates—0.94, 55 kPa replicates —1.0, Spearman correlation coefficient: 2 kPa replicates—0.90, 55 kPa replicates—0.90). ( B ) Total number of genes upregulated (≥log 2 - 2 fold) on the softer matrices—2 kPa (783 genes) and 55 kPa (649 genes), 670 and 536 genes were uniquely upregulated on 2 kPa and 55 kPa matrices respectively, while 113 genes were commonly upregulated on both the matrices. Selected genes from the maximally deregulated GO categories that are upregulated uniquely ( > log 2 - 10 fold) and commonly ( > log 2 -2 fold) on both the soft matrices are displayed. ( C ) Total number of genes downregulated (≥log 2 - 2 fold) on the softer matrices—2 kPa (872 genes) and 55 kPa (783 genes), 711 and 622 genes were uniquely downregulated on 2 kPa and 55 kPa matrices respectively, while 161 genes were commonly downregulated on both the matrices. Selected genes from the maximally deregulated GO categories that are downregulated uniquely ( > log 2 - 10 fold) and commonly ( > log 2 - 2 fold) on both the soft matrices are displayed. ( D ) Table depicting DNA content and Gene density of Chr. 1, 18 and 19. ( E ) Stacked bar graph depicting % deregulation (up and down) in cells on 2 kPa matrices, on all the chromosomes. Total number of ≥ log 2 - 2 fold deregulated genes on each chromosome were normalized to the total number of transcribing genes (FPKM > 1) on that chromosome. (Arrow) Chromosome 1 shows the maximum deregulation on 2 kPa (∼16.64%). (Red box) Chromosome 18 is amongst the chromosomes showing least transcriptional changes, while chromosome 19 is amongst the chromosomes showing high transcriptional deregulation. ( F ) Stacked bar graph depicting % deregulation (up and down) in cells on 55 kPa matrices, on all the chromosomes. Total number of ≥log 2 - 2 fold deregulated genes on each chromosome were normalized to the total number of transcribing genes (FPKM > 1) on that chromosome. (Red box) Chromosome 18 shows less transcriptional deregulation as compared to chromosome 19. ( G ) Experimental scheme. ( H ) Representation of fluorescence intensity quantification for each nucleus using line-scan analysis. ( I and J ) Representative mid-optical sections from confocal z -stack of DLD-1 cells immunostained for H3K4me3 on softer matrices (2 kPa and 55 kPa), glass coverslips and cells switched from 2 kPa to glass. Lower panel: zoom of single nucleus (J) Normalized average total fluorescence intensity of H3K4me3 under the above conditions (normalized to total nuclear surface area). ( K and L ) Representative mid-optical sections from confocal z-stack of DLD-1 cells immunostained for H3K27me3 on softer matrices (2 kPa and 55 kPa), glass coverslips and cells switched from 2 kPa to glass. Lower panel: zoom of single nucleus (L) Normalized average fluorescence intensity from line-scans across nuclei of H3K27me3 under the above conditions (J and L: n : number of nuclei, Pooled data from N = 2 independent biological replicates, Error bar: SEM, Mann–Whitney test). *** P

    Journal: Nucleic Acids Research

    Article Title: Emerin modulates spatial organization of chromosome territories in cells on softer matrices

    doi: 10.1093/nar/gky288

    Figure Lengend Snippet: Transcriptional deregulation and mislocalization of inactive histone marks is induced in cells exposed to softer matrices. ( A ) Experimental scheme. RNA sequencing was performed in two independent biological replicates (Pearson correlation coefficient: 2 kPa replicates—0.94, 55 kPa replicates —1.0, Spearman correlation coefficient: 2 kPa replicates—0.90, 55 kPa replicates—0.90). ( B ) Total number of genes upregulated (≥log 2 - 2 fold) on the softer matrices—2 kPa (783 genes) and 55 kPa (649 genes), 670 and 536 genes were uniquely upregulated on 2 kPa and 55 kPa matrices respectively, while 113 genes were commonly upregulated on both the matrices. Selected genes from the maximally deregulated GO categories that are upregulated uniquely ( > log 2 - 10 fold) and commonly ( > log 2 -2 fold) on both the soft matrices are displayed. ( C ) Total number of genes downregulated (≥log 2 - 2 fold) on the softer matrices—2 kPa (872 genes) and 55 kPa (783 genes), 711 and 622 genes were uniquely downregulated on 2 kPa and 55 kPa matrices respectively, while 161 genes were commonly downregulated on both the matrices. Selected genes from the maximally deregulated GO categories that are downregulated uniquely ( > log 2 - 10 fold) and commonly ( > log 2 - 2 fold) on both the soft matrices are displayed. ( D ) Table depicting DNA content and Gene density of Chr. 1, 18 and 19. ( E ) Stacked bar graph depicting % deregulation (up and down) in cells on 2 kPa matrices, on all the chromosomes. Total number of ≥ log 2 - 2 fold deregulated genes on each chromosome were normalized to the total number of transcribing genes (FPKM > 1) on that chromosome. (Arrow) Chromosome 1 shows the maximum deregulation on 2 kPa (∼16.64%). (Red box) Chromosome 18 is amongst the chromosomes showing least transcriptional changes, while chromosome 19 is amongst the chromosomes showing high transcriptional deregulation. ( F ) Stacked bar graph depicting % deregulation (up and down) in cells on 55 kPa matrices, on all the chromosomes. Total number of ≥log 2 - 2 fold deregulated genes on each chromosome were normalized to the total number of transcribing genes (FPKM > 1) on that chromosome. (Red box) Chromosome 18 shows less transcriptional deregulation as compared to chromosome 19. ( G ) Experimental scheme. ( H ) Representation of fluorescence intensity quantification for each nucleus using line-scan analysis. ( I and J ) Representative mid-optical sections from confocal z -stack of DLD-1 cells immunostained for H3K4me3 on softer matrices (2 kPa and 55 kPa), glass coverslips and cells switched from 2 kPa to glass. Lower panel: zoom of single nucleus (J) Normalized average total fluorescence intensity of H3K4me3 under the above conditions (normalized to total nuclear surface area). ( K and L ) Representative mid-optical sections from confocal z-stack of DLD-1 cells immunostained for H3K27me3 on softer matrices (2 kPa and 55 kPa), glass coverslips and cells switched from 2 kPa to glass. Lower panel: zoom of single nucleus (L) Normalized average fluorescence intensity from line-scans across nuclei of H3K27me3 under the above conditions (J and L: n : number of nuclei, Pooled data from N = 2 independent biological replicates, Error bar: SEM, Mann–Whitney test). *** P

    Article Snippet: Following primary antibodies—Rabbit anti-Lamin A (ab26300, 1:500), Rabbit anti-Lamin B1 (ab16048, 1:500), Mouse anti-Lamin B2 (ab8983, 1:400), Mouse anti-Emerin (SC-25284, 1:500), Rabbit anti-SUN1 (ab125770, 1:500), Rabbit anti-SUN2 (ab124916, 1:500), Rabbit anti-H3K27me3 (07-449, 1:500) and Rabbit anti-H3K4me3 (07-473, 1:500) were used.

    Techniques: RNA Sequencing Assay, Fluorescence, MANN-WHITNEY

    Relative enrichment of H3K4me3 and H3K27me3 for E. grandis homologs of transcription factors and signal peptides regulating vascular development and flowering in developing secondary xylem tissue. Blue bars, enrichment of H3K4 and H3K27 trimethylation (shown as N-terminal region of histone H3), represented as a signal of zero (no significant enrichment) to four bars (16-fold enrichment). Red bars, absolute RNA-seq expression levels (max FPKM value, 228) for roots, phloem, developing secondary xylem (DSX), shoots, young leaves (YL), mature leaves (ML) and flowers 41 , 42 .

    Journal: Scientific Reports

    Article Title: Integrated analysis and transcript abundance modelling of H3K4me3 and H3K27me3 in developing secondary xylem

    doi: 10.1038/s41598-017-03665-1

    Figure Lengend Snippet: Relative enrichment of H3K4me3 and H3K27me3 for E. grandis homologs of transcription factors and signal peptides regulating vascular development and flowering in developing secondary xylem tissue. Blue bars, enrichment of H3K4 and H3K27 trimethylation (shown as N-terminal region of histone H3), represented as a signal of zero (no significant enrichment) to four bars (16-fold enrichment). Red bars, absolute RNA-seq expression levels (max FPKM value, 228) for roots, phloem, developing secondary xylem (DSX), shoots, young leaves (YL), mature leaves (ML) and flowers 41 , 42 .

    Article Snippet: ChIP was performed with ~2 μg anti-H3K4me3 (Merck Millipore #07–473), anti-H3K27me3 (Merck Millipore, ABE44) or anti-RNA Pol II (Merck Millipore, #17–672) antibodies using the protocol described in Adli and Bernstein , without ChIP DNA amplification.

    Techniques: RNA Sequencing Assay, Expressing

    Association of H3K4me3 and H3K27me3 with gene expression levels and specificity. ( a ) Box plot of absolute expression values of genes from different epigenomic categories. The absolute expression profile for developing secondary xylem (far right) is shown for comparison. ( b ) Average per-base coverage of H3K4me3 (top) and H3K27me3 (bottom) ChIP-seq libraries around the transcription start site (TSS) of genes expressed at various levels in developing secondary xylem. ( c ) Tissue-specificity distribution of genes marked by different combinations of H3K4me3 and H3K27me3, as measured by the Shannon entropy index (calculated for genes with nonzero expression in at least one tissue). All pairwise combinations are statistically significant (Kolmogorov-Smirnov test, P

    Journal: Scientific Reports

    Article Title: Integrated analysis and transcript abundance modelling of H3K4me3 and H3K27me3 in developing secondary xylem

    doi: 10.1038/s41598-017-03665-1

    Figure Lengend Snippet: Association of H3K4me3 and H3K27me3 with gene expression levels and specificity. ( a ) Box plot of absolute expression values of genes from different epigenomic categories. The absolute expression profile for developing secondary xylem (far right) is shown for comparison. ( b ) Average per-base coverage of H3K4me3 (top) and H3K27me3 (bottom) ChIP-seq libraries around the transcription start site (TSS) of genes expressed at various levels in developing secondary xylem. ( c ) Tissue-specificity distribution of genes marked by different combinations of H3K4me3 and H3K27me3, as measured by the Shannon entropy index (calculated for genes with nonzero expression in at least one tissue). All pairwise combinations are statistically significant (Kolmogorov-Smirnov test, P

    Article Snippet: ChIP was performed with ~2 μg anti-H3K4me3 (Merck Millipore #07–473), anti-H3K27me3 (Merck Millipore, ABE44) or anti-RNA Pol II (Merck Millipore, #17–672) antibodies using the protocol described in Adli and Bernstein , without ChIP DNA amplification.

    Techniques: Expressing, Chromatin Immunoprecipitation

    Genomic distribution of H3K4me3 and H3K27me3 in E. grandis developing xylem. ( a ) Relative per-base coverage of H3K4me3, H3K27me3, RNA pol II and input control libraries across annotated transcription start sites (TSS). ( b ) Genomic features coinciding with H3K4me3 and H3K27me3 ChIP-seq peak summits. The proportion of each annotation in the genome is indicated on the far right for comparison.

    Journal: Scientific Reports

    Article Title: Integrated analysis and transcript abundance modelling of H3K4me3 and H3K27me3 in developing secondary xylem

    doi: 10.1038/s41598-017-03665-1

    Figure Lengend Snippet: Genomic distribution of H3K4me3 and H3K27me3 in E. grandis developing xylem. ( a ) Relative per-base coverage of H3K4me3, H3K27me3, RNA pol II and input control libraries across annotated transcription start sites (TSS). ( b ) Genomic features coinciding with H3K4me3 and H3K27me3 ChIP-seq peak summits. The proportion of each annotation in the genome is indicated on the far right for comparison.

    Article Snippet: ChIP was performed with ~2 μg anti-H3K4me3 (Merck Millipore #07–473), anti-H3K27me3 (Merck Millipore, ABE44) or anti-RNA Pol II (Merck Millipore, #17–672) antibodies using the protocol described in Adli and Bernstein , without ChIP DNA amplification.

    Techniques: Chromatin Immunoprecipitation

    Enrichment of H3K4me3 and H3K27me3 peak length clusters for unique biological processes. ( a ) Schematic representation of method. ( b ) Degree of enrichment for unique biological processes for each cluster. Selected terms associated with xylogenesis are indicated for the clusters where they occur. *Significantly enriched above expectation; binomial test, P

    Journal: Scientific Reports

    Article Title: Integrated analysis and transcript abundance modelling of H3K4me3 and H3K27me3 in developing secondary xylem

    doi: 10.1038/s41598-017-03665-1

    Figure Lengend Snippet: Enrichment of H3K4me3 and H3K27me3 peak length clusters for unique biological processes. ( a ) Schematic representation of method. ( b ) Degree of enrichment for unique biological processes for each cluster. Selected terms associated with xylogenesis are indicated for the clusters where they occur. *Significantly enriched above expectation; binomial test, P

    Article Snippet: ChIP was performed with ~2 μg anti-H3K4me3 (Merck Millipore #07–473), anti-H3K27me3 (Merck Millipore, ABE44) or anti-RNA Pol II (Merck Millipore, #17–672) antibodies using the protocol described in Adli and Bernstein , without ChIP DNA amplification.

    Techniques:

    PLOD2 promoter histone methylation is affected by TGFβ1 stimulation. A , qChIP against H3K4me3 from fibroblasts treated with TGFβ1 or vehicle for 48 h ( B ) qChIP on fibroblast treated with TGFβ1 or vehicle for 48 h, with antibodies against ASH1L. C , Western blot of fibroblasts transfected with esiRNA against ASH1L or control and treated with TGFβ1 or vehicle for 48 h. Blots were stained with antibodies against ASH1L, PLOD2 and YWAHZ as loading control. qChIP on fibroblasts treated with TGFβ1 or vehicle for 48 h, with antibodies against gene activation related ( D ) H3K79me2, and gene repression related modifications: ( E ) H3K9me3, ( F ) H3K27me3, and ( G ) H3K20me3. In all ChIP experiments normal rIgG was used as control and enrichment for PLOD2 −73/+50 was checked by qPCR, and normalized to input. Data are represented as mean ± S.E. ( n = 3), *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Procollagen Lysyl Hydroxylase 2 Expression Is Regulated by an Alternative Downstream Transforming Growth Factor β-1 Activation Mechanism *

    doi: 10.1074/jbc.M114.634311

    Figure Lengend Snippet: PLOD2 promoter histone methylation is affected by TGFβ1 stimulation. A , qChIP against H3K4me3 from fibroblasts treated with TGFβ1 or vehicle for 48 h ( B ) qChIP on fibroblast treated with TGFβ1 or vehicle for 48 h, with antibodies against ASH1L. C , Western blot of fibroblasts transfected with esiRNA against ASH1L or control and treated with TGFβ1 or vehicle for 48 h. Blots were stained with antibodies against ASH1L, PLOD2 and YWAHZ as loading control. qChIP on fibroblasts treated with TGFβ1 or vehicle for 48 h, with antibodies against gene activation related ( D ) H3K79me2, and gene repression related modifications: ( E ) H3K9me3, ( F ) H3K27me3, and ( G ) H3K20me3. In all ChIP experiments normal rIgG was used as control and enrichment for PLOD2 −73/+50 was checked by qPCR, and normalized to input. Data are represented as mean ± S.E. ( n = 3), *, p

    Article Snippet: Next, 40 μl Protein-G or -A Dynabeads® (Life Technologies) were coated with 5 μg antibodies per ChIP reaction; H3ac (Merck Millipore, Billerica, MA), H4ac (Merck Millipore), H3K4me3 (Merck Millipore), H3K9me3 (Abcam), H3K27me3 (Merk Millipore), H3K79me2 (Abcam), H4K20me3 (Abcam), CBP (Abcam), P300 (Merck Millipore), HDAC1 (Santa Cruz Biotechnology), HDAC2 (Santa Cruz Biotechnology), SP1 (Abcam), SMAD3 (Abcam), RNA pol II CTD repeat (phospho S5) (Abcam) and normal rIgG (Abcam) as control.

    Techniques: Methylation, Western Blot, Transfection, esiRNA, Staining, Activation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Depletion of Smad3 reduces PLOD2 promoter histone acetylation. Fibroblasts transfected with esiRNA against SMAD3 or control and treated with TGFβ1 for 48 h and used for qChIP with antibodies against: ( A ) H3ac, ( B ) H4ac, ( C ) H3K4me3, and ( D ) H3K9me3. Normal rIgG was used as control and enrichment for PLOD2 regions −73/+50 and −792/−657 was checked by qPCR, and normalized to input. Data are represented as mean ± S.E. ( n = 3), *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Procollagen Lysyl Hydroxylase 2 Expression Is Regulated by an Alternative Downstream Transforming Growth Factor β-1 Activation Mechanism *

    doi: 10.1074/jbc.M114.634311

    Figure Lengend Snippet: Depletion of Smad3 reduces PLOD2 promoter histone acetylation. Fibroblasts transfected with esiRNA against SMAD3 or control and treated with TGFβ1 for 48 h and used for qChIP with antibodies against: ( A ) H3ac, ( B ) H4ac, ( C ) H3K4me3, and ( D ) H3K9me3. Normal rIgG was used as control and enrichment for PLOD2 regions −73/+50 and −792/−657 was checked by qPCR, and normalized to input. Data are represented as mean ± S.E. ( n = 3), *, p

    Article Snippet: Next, 40 μl Protein-G or -A Dynabeads® (Life Technologies) were coated with 5 μg antibodies per ChIP reaction; H3ac (Merck Millipore, Billerica, MA), H4ac (Merck Millipore), H3K4me3 (Merck Millipore), H3K9me3 (Abcam), H3K27me3 (Merk Millipore), H3K79me2 (Abcam), H4K20me3 (Abcam), CBP (Abcam), P300 (Merck Millipore), HDAC1 (Santa Cruz Biotechnology), HDAC2 (Santa Cruz Biotechnology), SP1 (Abcam), SMAD3 (Abcam), RNA pol II CTD repeat (phospho S5) (Abcam) and normal rIgG (Abcam) as control.

    Techniques: Transfection, esiRNA, Real-time Polymerase Chain Reaction

    Lack of Ccl1 results in decreased global H3K4me3. The Fusarium wild type strains ( F. fujikuroi – FfWT, F. graminearum – FgWT) and the respective CCL1 deletion mutants ( F. fujikuroi – Δ ffccl1 , F. graminearum – Δ fgccl1 ) were grown for 3 days on solid complete medium. Whole protein extracts were subsequently isolated from lyophilized mycelia and roughly 15 μg of proteins were used for SDS-Page and western blotting. (A) The following primary antibodies were used for detection: H3 C-Term, H3K4me1, H3K4me2 as well as three different H3K4me3. (B) Coomassie staining was performed as an additional loading control.

    Journal: Frontiers in Microbiology

    Article Title: Lack of the COMPASS Component Ccl1 Reduces H3K4 Trimethylation Levels and Affects Transcription of Secondary Metabolite Genes in Two Plant–Pathogenic Fusarium Species

    doi: 10.3389/fmicb.2016.02144

    Figure Lengend Snippet: Lack of Ccl1 results in decreased global H3K4me3. The Fusarium wild type strains ( F. fujikuroi – FfWT, F. graminearum – FgWT) and the respective CCL1 deletion mutants ( F. fujikuroi – Δ ffccl1 , F. graminearum – Δ fgccl1 ) were grown for 3 days on solid complete medium. Whole protein extracts were subsequently isolated from lyophilized mycelia and roughly 15 μg of proteins were used for SDS-Page and western blotting. (A) The following primary antibodies were used for detection: H3 C-Term, H3K4me1, H3K4me2 as well as three different H3K4me3. (B) Coomassie staining was performed as an additional loading control.

    Article Snippet: The membrane was probed with H3 C-Term (Active Motif AM39163), H3K4me1 (Active Motif AM39297), H3K4me2 (Active Motif AM39141) as well as three different H3K4me3 (Active Motif AM39159, abcam ab8580, Millipore MP#07-473) primary antibodies and anti-rabbit (Sigma A0545) HRP conjugated secondary antibody.

    Techniques: Isolation, SDS Page, Western Blot, Staining

    Chromatin immunoprecipitation (ChIP) reveals distinct alterations in H3K4 dimethylation at SM gene clusters. The F. fujikuroi wild type strain (FfWT) and the CCL1 deletion mutant (Δ ffccl1 ) were grown in synthetic ICI medium with either 60 mM (fusarin) or 6 mM glutamine (bikaverin and gibberellic acid) for 3 days. Mycelium was crosslinked and used for ChIP analyses (Left ). In case of F. graminearum , the wild type (FgWT) and Δ fgccl1 were grown for 4 days on PDA (Right ). ChIP assays were conducted by using H3K9 acetylation (H3K9ac)-, H3K4me2- and H3K4me3-specific antibodies. Precipitated genomic DNA was quantified by quantitative real-time PCR using primer pairs located in the 5′ region of investigated SM cluster genes. For each SM cluster, two cluster genes were analyzed. Experiments were performed in biological and technical duplicates. The amount of precipitated DNA in the respective wild type strain was arbitrarily set to 1. Mean values and standard deviations are shown. PTMs, post-translational modifications.

    Journal: Frontiers in Microbiology

    Article Title: Lack of the COMPASS Component Ccl1 Reduces H3K4 Trimethylation Levels and Affects Transcription of Secondary Metabolite Genes in Two Plant–Pathogenic Fusarium Species

    doi: 10.3389/fmicb.2016.02144

    Figure Lengend Snippet: Chromatin immunoprecipitation (ChIP) reveals distinct alterations in H3K4 dimethylation at SM gene clusters. The F. fujikuroi wild type strain (FfWT) and the CCL1 deletion mutant (Δ ffccl1 ) were grown in synthetic ICI medium with either 60 mM (fusarin) or 6 mM glutamine (bikaverin and gibberellic acid) for 3 days. Mycelium was crosslinked and used for ChIP analyses (Left ). In case of F. graminearum , the wild type (FgWT) and Δ fgccl1 were grown for 4 days on PDA (Right ). ChIP assays were conducted by using H3K9 acetylation (H3K9ac)-, H3K4me2- and H3K4me3-specific antibodies. Precipitated genomic DNA was quantified by quantitative real-time PCR using primer pairs located in the 5′ region of investigated SM cluster genes. For each SM cluster, two cluster genes were analyzed. Experiments were performed in biological and technical duplicates. The amount of precipitated DNA in the respective wild type strain was arbitrarily set to 1. Mean values and standard deviations are shown. PTMs, post-translational modifications.

    Article Snippet: The membrane was probed with H3 C-Term (Active Motif AM39163), H3K4me1 (Active Motif AM39297), H3K4me2 (Active Motif AM39141) as well as three different H3K4me3 (Active Motif AM39159, abcam ab8580, Millipore MP#07-473) primary antibodies and anti-rabbit (Sigma A0545) HRP conjugated secondary antibody.

    Techniques: Chromatin Immunoprecipitation, Mutagenesis, Real-time Polymerase Chain Reaction

    Reduction of H3K4me3 does not affect expression of regulated genes a , Distribution of ASH2 binding in stable (red), regulated (blue) and silent genes (grey). b , H3K4me3 is strongly decreased in ash2 I1 mutant clones in WID. c , En immunostaining in WID (merged). The scale bar represents 20 μm. d , The levels of the stable gene En are reduced in mutant clones. e, GFP negative cells indicate ash2 I1 mutant cells in c and d . f , CycA immunostaining in WID (merged). The scale bar represents 20 μm. g, CycA is decreased in ash2 I1 mutant clones. h, GFP negative cells indicate ash2 I1 mutant cells in f and g . i, pdm2 fluorescence in situ hybridization in ash2 I1 mutant clones in WID (merged). The scale bar represents 20 μm. j , No changes in pdm2 expression are observed in ash2 I1 mutant clones. k, GFP negative cells indicate ash2 I1 mutant cells in i and j . l, Boss immunostaining in EID. The scale bar represents 20 μm. m, Optical cross-section (white line in l ) showing Boss in all R8 photoreceptor cells (merged). n , No changes in Boss expression are observed in ash2 I1 mutant clones. o, GFP negative cells indicate ash2 I1 mutant cells in m and n . p , Diagram summarizing the result in m – o . Green cells express the wild-type ash2 allele and black cells correspond to homozygous ash2 I1 mutant cells. Boss (magenta cap) localizes in the apical side of R8.

    Journal: Nature genetics

    Article Title: Absence of canonical active chromatin marks in developmentally regulated genes

    doi: 10.1038/ng.3381

    Figure Lengend Snippet: Reduction of H3K4me3 does not affect expression of regulated genes a , Distribution of ASH2 binding in stable (red), regulated (blue) and silent genes (grey). b , H3K4me3 is strongly decreased in ash2 I1 mutant clones in WID. c , En immunostaining in WID (merged). The scale bar represents 20 μm. d , The levels of the stable gene En are reduced in mutant clones. e, GFP negative cells indicate ash2 I1 mutant cells in c and d . f , CycA immunostaining in WID (merged). The scale bar represents 20 μm. g, CycA is decreased in ash2 I1 mutant clones. h, GFP negative cells indicate ash2 I1 mutant cells in f and g . i, pdm2 fluorescence in situ hybridization in ash2 I1 mutant clones in WID (merged). The scale bar represents 20 μm. j , No changes in pdm2 expression are observed in ash2 I1 mutant clones. k, GFP negative cells indicate ash2 I1 mutant cells in i and j . l, Boss immunostaining in EID. The scale bar represents 20 μm. m, Optical cross-section (white line in l ) showing Boss in all R8 photoreceptor cells (merged). n , No changes in Boss expression are observed in ash2 I1 mutant clones. o, GFP negative cells indicate ash2 I1 mutant cells in m and n . p , Diagram summarizing the result in m – o . Green cells express the wild-type ash2 allele and black cells correspond to homozygous ash2 I1 mutant cells. Boss (magenta cap) localizes in the apical side of R8.

    Article Snippet: The antibodies used for ChIP were: H3 (Abcam/ab1791); H3K4me3 (Abcam/ab8580) (Millipore-Upstate/07-473), H3K9ac (Abcam/ab4441), H3K36me3 (Abcam/ab9050), H3K4me1 (Diagenode/CS-037-100), H3K27ac (Abcam/ab4729) and H3K27me3 (Upstate-Millipore/07-449).

    Techniques: Expressing, Binding Assay, Mutagenesis, Clone Assay, Immunostaining, Fluorescence, In Situ Hybridization

    Gene expression and histone modifications in regulated broadly-expressed and stable tissue-specific genes at third instar larvae a, Diagrams of developmentally regulated genes broadly-expressed across multiple tissues at third instar-larvae L3 (left panel), and stable genes expressed in only one tissue at L3 (right panel). b, Gene expression levels at L3 measured by whole organism RNASeq (left panel). The number of genes in each category is given under the boxplots. The bottom and top of the boxes are the first and third quartiles, and the line within, the median. The whiskers denote the interval within 1.5 times the IQR from the median. Outliers are plotted as dots. Validation by qPCR of the expression at L3 of regulated broadly-expressed genes compared to a stable gene ( Bmcp ) and a silent gene ( CG5367 ) (right panel). Error bars represent the Standard Error of the Mean (SEM) from three independent replicates. c, Levels of H3K4me3, H3K9ac, H3K4me1 and H3K27ac on whole L3 individuals. The seven regulated genes broadly-expressed at L3 are depicted as red dots within the boxplots. P-values were computed using the Wilcoxon test (two-sided). d, Validation by individual ChIPs and qPCR of H3K4me3 and H3K9ac in regulated genes broadly-expressed at L3. H3K4me3 and H3K9ac ChIPs are represented as enrichment of the marks over the silent gene ( CG5367 ). Error bars represent the SEM from three independent replicates.

    Journal: Nature genetics

    Article Title: Absence of canonical active chromatin marks in developmentally regulated genes

    doi: 10.1038/ng.3381

    Figure Lengend Snippet: Gene expression and histone modifications in regulated broadly-expressed and stable tissue-specific genes at third instar larvae a, Diagrams of developmentally regulated genes broadly-expressed across multiple tissues at third instar-larvae L3 (left panel), and stable genes expressed in only one tissue at L3 (right panel). b, Gene expression levels at L3 measured by whole organism RNASeq (left panel). The number of genes in each category is given under the boxplots. The bottom and top of the boxes are the first and third quartiles, and the line within, the median. The whiskers denote the interval within 1.5 times the IQR from the median. Outliers are plotted as dots. Validation by qPCR of the expression at L3 of regulated broadly-expressed genes compared to a stable gene ( Bmcp ) and a silent gene ( CG5367 ) (right panel). Error bars represent the Standard Error of the Mean (SEM) from three independent replicates. c, Levels of H3K4me3, H3K9ac, H3K4me1 and H3K27ac on whole L3 individuals. The seven regulated genes broadly-expressed at L3 are depicted as red dots within the boxplots. P-values were computed using the Wilcoxon test (two-sided). d, Validation by individual ChIPs and qPCR of H3K4me3 and H3K9ac in regulated genes broadly-expressed at L3. H3K4me3 and H3K9ac ChIPs are represented as enrichment of the marks over the silent gene ( CG5367 ). Error bars represent the SEM from three independent replicates.

    Article Snippet: The antibodies used for ChIP were: H3 (Abcam/ab1791); H3K4me3 (Abcam/ab8580) (Millipore-Upstate/07-473), H3K9ac (Abcam/ab4441), H3K36me3 (Abcam/ab9050), H3K4me1 (Diagenode/CS-037-100), H3K27ac (Abcam/ab4729) and H3K27me3 (Upstate-Millipore/07-449).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Association between histone modifications and transcription stability in metazoans a, Scatterplot of H3K4me3 levels at the time point of highest expression during fly development and transcriptional stability measured as the coefficient of variation of gene expression across time points. The correlation is computed as the partial correlation given gene expression. b, Partial correlations between active marks and transcription stability (the coefficient of variation). Correlations are computed controlling for gene expression. All correlations are statistically significant (p-value

    Journal: Nature genetics

    Article Title: Absence of canonical active chromatin marks in developmentally regulated genes

    doi: 10.1038/ng.3381

    Figure Lengend Snippet: Association between histone modifications and transcription stability in metazoans a, Scatterplot of H3K4me3 levels at the time point of highest expression during fly development and transcriptional stability measured as the coefficient of variation of gene expression across time points. The correlation is computed as the partial correlation given gene expression. b, Partial correlations between active marks and transcription stability (the coefficient of variation). Correlations are computed controlling for gene expression. All correlations are statistically significant (p-value

    Article Snippet: The antibodies used for ChIP were: H3 (Abcam/ab1791); H3K4me3 (Abcam/ab8580) (Millipore-Upstate/07-473), H3K9ac (Abcam/ab4441), H3K36me3 (Abcam/ab9050), H3K4me1 (Diagenode/CS-037-100), H3K27ac (Abcam/ab4729) and H3K27me3 (Upstate-Millipore/07-449).

    Techniques: Expressing, Significance Assay

    Active transcription of pdm2 without chromatin modifications a, Expression of pdm2 in WID (left panel) and EID (middle panel) labeled with a pdm2 -specific probe. The gene is only expressed in the wing pouch of the WID, highlighted in green. The scale bars represent 100 μm. b, Expression of pdm2 in sorted cells analyzed by qPCR. Gene expression is normalized by the control gene crm . Error bars represent the SEM from three biological replicates. c , ChIP analysis of H3K4me3, H3K36me3 and of negative controls without antibody on sorted cells. ChIPs are represented as enrichment of the marks over a silent gene non-marked with H3K4me3 and H3K36me3 ( CG10013 ). Crm is used as positive control for these modifications. Error bars represent the SEM from at least three biological replicates. P-values were computed using the Student’s t-test (two-sided). d , Newly transcribed RNA of GFP-sorted cells. Nascent RNA is normalized by the control gene crm . Error bars represent the SEM of four biological replicates. e, ChIP analysis of H3K27me3 and of negative controls without antibody on sorted cells. H3K27me3 ChIPs are represented as enrichment of the mark over a constitutively expressed gene non-marked with H3K27me3 ( RpL32 ). Abd-B is used as positive control for this modification. Error bars represent the SEM from at least three biological replicates.

    Journal: Nature genetics

    Article Title: Absence of canonical active chromatin marks in developmentally regulated genes

    doi: 10.1038/ng.3381

    Figure Lengend Snippet: Active transcription of pdm2 without chromatin modifications a, Expression of pdm2 in WID (left panel) and EID (middle panel) labeled with a pdm2 -specific probe. The gene is only expressed in the wing pouch of the WID, highlighted in green. The scale bars represent 100 μm. b, Expression of pdm2 in sorted cells analyzed by qPCR. Gene expression is normalized by the control gene crm . Error bars represent the SEM from three biological replicates. c , ChIP analysis of H3K4me3, H3K36me3 and of negative controls without antibody on sorted cells. ChIPs are represented as enrichment of the marks over a silent gene non-marked with H3K4me3 and H3K36me3 ( CG10013 ). Crm is used as positive control for these modifications. Error bars represent the SEM from at least three biological replicates. P-values were computed using the Student’s t-test (two-sided). d , Newly transcribed RNA of GFP-sorted cells. Nascent RNA is normalized by the control gene crm . Error bars represent the SEM of four biological replicates. e, ChIP analysis of H3K27me3 and of negative controls without antibody on sorted cells. H3K27me3 ChIPs are represented as enrichment of the mark over a constitutively expressed gene non-marked with H3K27me3 ( RpL32 ). Abd-B is used as positive control for this modification. Error bars represent the SEM from at least three biological replicates.

    Article Snippet: The antibodies used for ChIP were: H3 (Abcam/ab1791); H3K4me3 (Abcam/ab8580) (Millipore-Upstate/07-473), H3K9ac (Abcam/ab4441), H3K36me3 (Abcam/ab9050), H3K4me1 (Diagenode/CS-037-100), H3K27ac (Abcam/ab4729) and H3K27me3 (Upstate-Millipore/07-449).

    Techniques: Expressing, Labeling, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Positive Control, Modification

    Distribution of histone modification levels in stable, regulated and silent genes during fly development a , Expression of stable, regulated, and silent genes during fly development at the time point of maximum expression for each gene. Gene expression was computed as FPKMs by the modENCODE consortium. The bottom and top of the boxes are the first and third quartiles, and the line within, the median. The whiskers denote the interval within 1.5 times the Inter Quartile Range (IQR) from the median. Outliers are plotted as dots. b, Normalized levels of H3K4me3, H3K9ac, H3K4me1 and H3K27ac at the time point of maximum expression during D. melanogaster development. These values represent the maximum height of the ChIPSeq peak within the gene body. P-values were computed using the Wilcoxon text (two-sided). c , Profiles of H3K4me3 during the 12 fly developmental time points in CG8636 , a gene stably expressed during fly development, and CG16733 , a pupa-specific gene. The expression (measured as FPKMs) along these points for the two genes is given on the left. d , Levels of H3K27me3 and H3K9me3 at the time point of maximum expression, computed as the average height of the ChIPSeq signal within the gene body, in stable, regulated and silent genes.

    Journal: Nature genetics

    Article Title: Absence of canonical active chromatin marks in developmentally regulated genes

    doi: 10.1038/ng.3381

    Figure Lengend Snippet: Distribution of histone modification levels in stable, regulated and silent genes during fly development a , Expression of stable, regulated, and silent genes during fly development at the time point of maximum expression for each gene. Gene expression was computed as FPKMs by the modENCODE consortium. The bottom and top of the boxes are the first and third quartiles, and the line within, the median. The whiskers denote the interval within 1.5 times the Inter Quartile Range (IQR) from the median. Outliers are plotted as dots. b, Normalized levels of H3K4me3, H3K9ac, H3K4me1 and H3K27ac at the time point of maximum expression during D. melanogaster development. These values represent the maximum height of the ChIPSeq peak within the gene body. P-values were computed using the Wilcoxon text (two-sided). c , Profiles of H3K4me3 during the 12 fly developmental time points in CG8636 , a gene stably expressed during fly development, and CG16733 , a pupa-specific gene. The expression (measured as FPKMs) along these points for the two genes is given on the left. d , Levels of H3K27me3 and H3K9me3 at the time point of maximum expression, computed as the average height of the ChIPSeq signal within the gene body, in stable, regulated and silent genes.

    Article Snippet: The antibodies used for ChIP were: H3 (Abcam/ab1791); H3K4me3 (Abcam/ab8580) (Millipore-Upstate/07-473), H3K9ac (Abcam/ab4441), H3K36me3 (Abcam/ab9050), H3K4me1 (Diagenode/CS-037-100), H3K27ac (Abcam/ab4729) and H3K27me3 (Upstate-Millipore/07-449).

    Techniques: Modification, Expressing, Stable Transfection