Structured Review

Millipore lysine 4
Chromatin immunoprecipitation using antibodies specific for methylated, unmodified and acetylated histones . Representative results of quantified DNA derived from unstimulated (basal) and 5-aza-CdR- or TSA-stimulated DU145 and MCF-7 cells immunoprecipitated by antibodies specific for methylated histones (left diagrams) as well as for unmodified and acetylated histones (right diagrams). Examples of PTEN in DU145 cells (A, B), CD44 in MCF-7 cells (C, D), GLIPR1 in DU145 cells (E, F) and Cyclin D2 in DU145 cells (G, H) are shown. All values obtained were normalized and referred to 100% of the input DNA. IgG, negative control; H3K9, Lysine 9 of histone H3; H3K4, <t>Lysine</t> 4 of histone H3; H4K20, Lysine 20 of histone H4; H2A, histone H2A; H2B, histone H2B; me, mono-methylated; me2, dimethylated; me3, trimethylated; Ac, acetylated.
Lysine 4, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Promoter- and cell-specific epigenetic regulation of CD44, Cyclin D2, GLIPR1 and PTEN by Methyl-CpG binding proteins and histone modifications"

Article Title: Promoter- and cell-specific epigenetic regulation of CD44, Cyclin D2, GLIPR1 and PTEN by Methyl-CpG binding proteins and histone modifications

Journal: BMC Cancer

doi: 10.1186/1471-2407-10-297

Chromatin immunoprecipitation using antibodies specific for methylated, unmodified and acetylated histones . Representative results of quantified DNA derived from unstimulated (basal) and 5-aza-CdR- or TSA-stimulated DU145 and MCF-7 cells immunoprecipitated by antibodies specific for methylated histones (left diagrams) as well as for unmodified and acetylated histones (right diagrams). Examples of PTEN in DU145 cells (A, B), CD44 in MCF-7 cells (C, D), GLIPR1 in DU145 cells (E, F) and Cyclin D2 in DU145 cells (G, H) are shown. All values obtained were normalized and referred to 100% of the input DNA. IgG, negative control; H3K9, Lysine 9 of histone H3; H3K4, Lysine 4 of histone H3; H4K20, Lysine 20 of histone H4; H2A, histone H2A; H2B, histone H2B; me, mono-methylated; me2, dimethylated; me3, trimethylated; Ac, acetylated.
Figure Legend Snippet: Chromatin immunoprecipitation using antibodies specific for methylated, unmodified and acetylated histones . Representative results of quantified DNA derived from unstimulated (basal) and 5-aza-CdR- or TSA-stimulated DU145 and MCF-7 cells immunoprecipitated by antibodies specific for methylated histones (left diagrams) as well as for unmodified and acetylated histones (right diagrams). Examples of PTEN in DU145 cells (A, B), CD44 in MCF-7 cells (C, D), GLIPR1 in DU145 cells (E, F) and Cyclin D2 in DU145 cells (G, H) are shown. All values obtained were normalized and referred to 100% of the input DNA. IgG, negative control; H3K9, Lysine 9 of histone H3; H3K4, Lysine 4 of histone H3; H4K20, Lysine 20 of histone H4; H2A, histone H2A; H2B, histone H2B; me, mono-methylated; me2, dimethylated; me3, trimethylated; Ac, acetylated.

Techniques Used: Chromatin Immunoprecipitation, Methylation, Derivative Assay, Immunoprecipitation, Negative Control

2) Product Images from "Promoter- and cell-specific epigenetic regulation of CD44, Cyclin D2, GLIPR1 and PTEN by Methyl-CpG binding proteins and histone modifications"

Article Title: Promoter- and cell-specific epigenetic regulation of CD44, Cyclin D2, GLIPR1 and PTEN by Methyl-CpG binding proteins and histone modifications

Journal: BMC Cancer

doi: 10.1186/1471-2407-10-297

Chromatin immunoprecipitation using antibodies specific for methylated, unmodified and acetylated histones . Representative results of quantified DNA derived from unstimulated (basal) and 5-aza-CdR- or TSA-stimulated DU145 and MCF-7 cells immunoprecipitated by antibodies specific for methylated histones (left diagrams) as well as for unmodified and acetylated histones (right diagrams). Examples of PTEN in DU145 cells (A, B), CD44 in MCF-7 cells (C, D), GLIPR1 in DU145 cells (E, F) and Cyclin D2 in DU145 cells (G, H) are shown. All values obtained were normalized and referred to 100% of the input DNA. IgG, negative control; H3K9, Lysine 9 of histone H3; H3K4, Lysine 4 of histone H3; H4K20, Lysine 20 of histone H4; H2A, histone H2A; H2B, histone H2B; me, mono-methylated; me2, dimethylated; me3, trimethylated; Ac, acetylated.
Figure Legend Snippet: Chromatin immunoprecipitation using antibodies specific for methylated, unmodified and acetylated histones . Representative results of quantified DNA derived from unstimulated (basal) and 5-aza-CdR- or TSA-stimulated DU145 and MCF-7 cells immunoprecipitated by antibodies specific for methylated histones (left diagrams) as well as for unmodified and acetylated histones (right diagrams). Examples of PTEN in DU145 cells (A, B), CD44 in MCF-7 cells (C, D), GLIPR1 in DU145 cells (E, F) and Cyclin D2 in DU145 cells (G, H) are shown. All values obtained were normalized and referred to 100% of the input DNA. IgG, negative control; H3K9, Lysine 9 of histone H3; H3K4, Lysine 4 of histone H3; H4K20, Lysine 20 of histone H4; H2A, histone H2A; H2B, histone H2B; me, mono-methylated; me2, dimethylated; me3, trimethylated; Ac, acetylated.

Techniques Used: Chromatin Immunoprecipitation, Methylation, Derivative Assay, Immunoprecipitation, Negative Control

3) Product Images from "Promoter- and cell-specific epigenetic regulation of CD44, Cyclin D2, GLIPR1 and PTEN by Methyl-CpG binding proteins and histone modifications"

Article Title: Promoter- and cell-specific epigenetic regulation of CD44, Cyclin D2, GLIPR1 and PTEN by Methyl-CpG binding proteins and histone modifications

Journal: BMC Cancer

doi: 10.1186/1471-2407-10-297

Chromatin immunoprecipitation using antibodies specific for methylated, unmodified and acetylated histones . Representative results of quantified DNA derived from unstimulated (basal) and 5-aza-CdR- or TSA-stimulated DU145 and MCF-7 cells immunoprecipitated by antibodies specific for methylated histones (left diagrams) as well as for unmodified and acetylated histones (right diagrams). Examples of PTEN in DU145 cells (A, B), CD44 in MCF-7 cells (C, D), GLIPR1 in DU145 cells (E, F) and Cyclin D2 in DU145 cells (G, H) are shown. All values obtained were normalized and referred to 100% of the input DNA. IgG, negative control; H3K9, Lysine 9 of histone H3; H3K4, Lysine 4 of histone H3; H4K20, Lysine 20 of histone H4; H2A, histone H2A; H2B, histone H2B; me, mono-methylated; me2, dimethylated; me3, trimethylated; Ac, acetylated.
Figure Legend Snippet: Chromatin immunoprecipitation using antibodies specific for methylated, unmodified and acetylated histones . Representative results of quantified DNA derived from unstimulated (basal) and 5-aza-CdR- or TSA-stimulated DU145 and MCF-7 cells immunoprecipitated by antibodies specific for methylated histones (left diagrams) as well as for unmodified and acetylated histones (right diagrams). Examples of PTEN in DU145 cells (A, B), CD44 in MCF-7 cells (C, D), GLIPR1 in DU145 cells (E, F) and Cyclin D2 in DU145 cells (G, H) are shown. All values obtained were normalized and referred to 100% of the input DNA. IgG, negative control; H3K9, Lysine 9 of histone H3; H3K4, Lysine 4 of histone H3; H4K20, Lysine 20 of histone H4; H2A, histone H2A; H2B, histone H2B; me, mono-methylated; me2, dimethylated; me3, trimethylated; Ac, acetylated.

Techniques Used: Chromatin Immunoprecipitation, Methylation, Derivative Assay, Immunoprecipitation, Negative Control

4) Product Images from "Activation of the B Cell Antigen Receptor Triggers Reactivation of Latent Kaposi's Sarcoma-Associated Herpesvirus in B Cells"

Article Title: Activation of the B Cell Antigen Receptor Triggers Reactivation of Latent Kaposi's Sarcoma-Associated Herpesvirus in B Cells

Journal: Journal of Virology

doi: 10.1128/JVI.00506-13

Comparison of global histone modification patterns of KSHV genomes in BrK.219 and PEL cells. Global patterns of histone H3 trimethylated at lysine 4 (H3K4-me3), lysine 9 (H3K9-me3), or lysine 27 (H3K27-me3) or acetylated at lysine 9 and/or lysine 14 (H3K27-me3
Figure Legend Snippet: Comparison of global histone modification patterns of KSHV genomes in BrK.219 and PEL cells. Global patterns of histone H3 trimethylated at lysine 4 (H3K4-me3), lysine 9 (H3K9-me3), or lysine 27 (H3K27-me3) or acetylated at lysine 9 and/or lysine 14 (H3K27-me3

Techniques Used: Modification

5) Product Images from "Ontogenic Expression of Hepatic Ahr mRNA is associated with Histone H3K4 Di-methylation during Mouse Liver Development"

Article Title: Ontogenic Expression of Hepatic Ahr mRNA is associated with Histone H3K4 Di-methylation during Mouse Liver Development

Journal: Toxicology letters

doi: 10.1016/j.toxlet.2009.05.017

Di-methylation of histone H3 at lysine-4 (H3K4Me2) at the Ahr gene locus during mouse liver development. Upper panel: browser view of fold changes of histone H3K4Me2 at the Ahr gene locus at day -2, 1, 5, and 45 of age (equal amount of pooled samples
Figure Legend Snippet: Di-methylation of histone H3 at lysine-4 (H3K4Me2) at the Ahr gene locus during mouse liver development. Upper panel: browser view of fold changes of histone H3K4Me2 at the Ahr gene locus at day -2, 1, 5, and 45 of age (equal amount of pooled samples

Techniques Used: Methylation

Related Articles

Modification:

Article Title: Tissue-Specific Epigenetic Modifications in Root Apical Meristem Cells of Hordeum vulgare
Article Snippet: .. Briefly, the following rabbit monoclonal and polyclonal antibodies against modified histones and DNA were used: anti-acetyl histone H4 at lysine 5 (1∶100; Millipore, Cat. no. 04-118), anti-dimethyl histone H3 at lysine 4 (1∶100 dilution in 1% BSA in PBS; Millipore, Cat. no. 07-030 and Cat. no. 07-790), anti-dimethyl histone H3 at lysine 9 (1∶100; Upstate, Cat. no. 05-768 and 07-212), anti-5-methyl-cytosine (1∶300, Abcam, Cat. no. ab73938). .. As secondary antibodies, Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, Molecular Probes Cat. no. A-11008) and Alexa Fluor 488 goat anti-mouse IgG (Invitrogen, Molecular Probes, Cat. no. A-11001) were applied.

Incubation:

Article Title: Desensitization and Incomplete Recovery of Hepatic Target Genes After Chronic Thyroid Hormone Treatment and Withdrawal in Male Adult Mice
Article Snippet: .. Samples were precleared and incubated with antibodies against histone H3 acetylated at lysines 9 and 14 (H3K9/K14ac; number 06-599; Millipore), histone H4 pan-acetylated at lysines 5, 8, 12, and 16 (Pan-H4ac; number 06-598; Millipore), histone H3 trimethylated at lysine 4 (H3K4me3; number 07-473; Millipore), or normal rabbit IgG (sc2027; Santa Cruz Biotechnology) overnight at 4°C ( ). ..

Article Title: Elucidating the molecular bases of epigenetic inheritance in non-model invertebrates: the case of the root-knot nematode Meloidogyne incognita
Article Snippet: .. The membrane was blocked overnight at 4°C in blocking buffer and incubated with one of the 12 following commercial antibodies (1μg/μl): Anti-histone H3 (H3, abcam ab1791 and Active Motif 39164); Anti-Histone H3 dimethylated at lysine 4 (H3K4Me2, Abcam ab32356) or at lysine 36 (H3K36Me2, Abcam ab9049); Anti-Histone H3 trimethylated at lysine 4 (H3K4Me3, Millipore 04-745 and Abcam ab8580), at lysine 9 (H3K9Me3, Abcam ab8898 and Upstate) or at lysine 27 (H3K27Me3, Diagenode pAB-069-050); Anti-Histone H3 acetylated at lysine 9 (H3K9Ac, Upstate); Anti-Histone H4 trimethylated at lysine 20 (H4K20Me3, abcam ab9053) and Anti-hyperacetylated Histone H4 (H4PentaAc, Upstate). .. Bands were revealed by Enhanced Chemical Luminescence (ECL Pierce) and direct exposure to X-ray film (Amersham).

Chromatin Immunoprecipitation:

Article Title: Histone Deacetylase Inhibition Activates Transgene Expression from Integration-Defective Lentiviral Vectors in Dividing and Non-Dividing Cells
Article Snippet: .. The antibodies used for the ChIP analysis were raised against histone 3 (H3; Millipore) or against H3 protein variants acetylated at lysines 9 and 14 (H3K9/K14ac; Epigentek) or tri-methylated at lysine 4 (H3K4me3; Millipore). .. The resulting immunoprecipitated DNA and corresponding input DNA were quantified by qPCR using the primers and reaction conditions specified in and the general cycling parameters given under the section “Vector titrations.” The qPCR data were presented as percent input and were calculated according to the following formula (%) (where Ct is the cycle threshold): 2^(-[Ct(ChIP)-Ct(Input)])*100*DNA input dilution factor, where Ct(ChIP) is the Ct value of the immunoprecipitated DNA using the antibody of interest and Ct(Input) is the Ct value of the input DNA.

Article Title: CRISPR/Transposon gene integration (CRITGI) can manage gene expression in a retrotransposon-dependent manner
Article Snippet: .. Monoclonal antibody to recognize trimethylated histone H3 on lysine 4 (H3-K4 me3) (04-745, Merk Millipore, Burlington, MA, USA) was employed for ChIP . .. The LightCycler 480 SYBR Green I Master (Roche Life Science, Penzberg, Germany) was used to calculate the percent of input for ChIP for histone H3-K4 me3.

Blocking Assay:

Article Title: Elucidating the molecular bases of epigenetic inheritance in non-model invertebrates: the case of the root-knot nematode Meloidogyne incognita
Article Snippet: .. The membrane was blocked overnight at 4°C in blocking buffer and incubated with one of the 12 following commercial antibodies (1μg/μl): Anti-histone H3 (H3, abcam ab1791 and Active Motif 39164); Anti-Histone H3 dimethylated at lysine 4 (H3K4Me2, Abcam ab32356) or at lysine 36 (H3K36Me2, Abcam ab9049); Anti-Histone H3 trimethylated at lysine 4 (H3K4Me3, Millipore 04-745 and Abcam ab8580), at lysine 9 (H3K9Me3, Abcam ab8898 and Upstate) or at lysine 27 (H3K27Me3, Diagenode pAB-069-050); Anti-Histone H3 acetylated at lysine 9 (H3K9Ac, Upstate); Anti-Histone H4 trimethylated at lysine 20 (H4K20Me3, abcam ab9053) and Anti-hyperacetylated Histone H4 (H4PentaAc, Upstate). .. Bands were revealed by Enhanced Chemical Luminescence (ECL Pierce) and direct exposure to X-ray film (Amersham).

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  • 99
    Millipore anti trimethyl histone h3
    <t>Histone</t> modifications mark differential alleles. (A) ChIP assay in HUVECs shows distributions of modified <t>histones</t> (K9 acetylated H3, K4 di- and trimethylated H3, K27 trimethylated H3, K9 dimethylated H3, and K36 trimethylated H3) along SHC1 . Error bars indicate means ± standard deviations (SD) of three different ChIP experiments. (B) ApoI digestion efficiency in allele-specific ChIP assay. (C) Quantitative, nanofluidic, and allele-specific digital PCR was used to evaluate the percentages of genotypes of ChIP products in HUVECs generating p52 Shc from the G allele and p66 Shc from the A allele (left) and HUVECs generating p52 Shc from the A allele and p66 Shc from the G allele (right). Error bars indicate means ± SD for three different ChIP experiments.
    Anti Trimethyl Histone H3, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti trimethyl histone h3/product/Millipore
    Average 99 stars, based on 21 article reviews
    Price from $9.99 to $1999.99
    anti trimethyl histone h3 - by Bioz Stars, 2020-09
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    85
    Millipore dynamin inhibitory compounds dynasore
    Lamellipodia formation and dynamics in pancreatic tumor cells are dependent upon Dyn2 expression and phosphorylation (a) Western blot analysis of stable BxPC-3 cell lines expressing either GFP alone, Dyn2-GFP, or Dyn2Y(231,597)F-GFP. (b-d) Control BxPC-3 cells stained for cortactin (b) grow in tight clusters and appear non-motile, while stable lines expressing Dyn2-GFP (c) exhibit numerous dynamic lamellipodia. (d) In comparison, mutant Dyn2Y(231,597)F-GFP expressing cells appear loosely clustered with some motile morphology. (c-d) show GFP fluorescence of the Dyn2-GFP and Dyn2Y(231,597)F-GFP. (e) Kymographs generated from movies of the lamellipodia of BxPC-3 stable cells showing the rapid protrusion rate of lamellipodia in cells expressing WT Dyn2-GFP, relative to cells expressing GFP or Dyn2Y(231/597)F -GFP. (f) Average lamellipodia protrusion speed calculated from more than 11 cells per cell line based on collected kymographs shown in (e). (g) Western blot on lysates from BxPC-3-GFP cells treated with non-targeting siRNA (NTsi) or Dyn2 siRNA (D2si). Kymographs (i) and quantitation (i) showing reduced lamellipodia extension following Dyn2 knockdown. Kymographs (j) and quantitation (k) showing lamellipodia extension of BxPC-3-GFP cells following treatment with DMSO or <t>dynamin</t> inhibitors <t>Dynasore</t> (80 μM) or MiTMAB (20 μM). Error bars represent S.E.M. Student’s t-test was used for statistics (** represents p
    Dynamin Inhibitory Compounds Dynasore, 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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    91
    Millipore lysine 4
    Chromatin immunoprecipitation using antibodies specific for methylated, unmodified and acetylated histones . Representative results of quantified DNA derived from unstimulated (basal) and 5-aza-CdR- or TSA-stimulated DU145 and MCF-7 cells immunoprecipitated by antibodies specific for methylated histones (left diagrams) as well as for unmodified and acetylated histones (right diagrams). Examples of PTEN in DU145 cells (A, B), CD44 in MCF-7 cells (C, D), GLIPR1 in DU145 cells (E, F) and Cyclin D2 in DU145 cells (G, H) are shown. All values obtained were normalized and referred to 100% of the input DNA. IgG, negative control; H3K9, Lysine 9 of histone H3; H3K4, <t>Lysine</t> 4 of histone H3; H4K20, Lysine 20 of histone H4; H2A, histone H2A; H2B, histone H2B; me, mono-methylated; me2, dimethylated; me3, trimethylated; Ac, acetylated.
    Lysine 4, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lysine 4/product/Millipore
    Average 91 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    lysine 4 - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    Image Search Results


    Histone modifications mark differential alleles. (A) ChIP assay in HUVECs shows distributions of modified histones (K9 acetylated H3, K4 di- and trimethylated H3, K27 trimethylated H3, K9 dimethylated H3, and K36 trimethylated H3) along SHC1 . Error bars indicate means ± standard deviations (SD) of three different ChIP experiments. (B) ApoI digestion efficiency in allele-specific ChIP assay. (C) Quantitative, nanofluidic, and allele-specific digital PCR was used to evaluate the percentages of genotypes of ChIP products in HUVECs generating p52 Shc from the G allele and p66 Shc from the A allele (left) and HUVECs generating p52 Shc from the A allele and p66 Shc from the G allele (right). Error bars indicate means ± SD for three different ChIP experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Heritable, Allele-Specific Chromosomal Looping between Tandem Promoters Specifies Promoter Usage of SHC1

    doi: 10.1128/MCB.00658-17

    Figure Lengend Snippet: Histone modifications mark differential alleles. (A) ChIP assay in HUVECs shows distributions of modified histones (K9 acetylated H3, K4 di- and trimethylated H3, K27 trimethylated H3, K9 dimethylated H3, and K36 trimethylated H3) along SHC1 . Error bars indicate means ± standard deviations (SD) of three different ChIP experiments. (B) ApoI digestion efficiency in allele-specific ChIP assay. (C) Quantitative, nanofluidic, and allele-specific digital PCR was used to evaluate the percentages of genotypes of ChIP products in HUVECs generating p52 Shc from the G allele and p66 Shc from the A allele (left) and HUVECs generating p52 Shc from the A allele and p66 Shc from the G allele (right). Error bars indicate means ± SD for three different ChIP experiments.

    Article Snippet: Samples were then diluted into NET buffer (50 mM Tris [pH7.4], 150 mM NaCl, 5 mM EDTA, 0.5% NP-40), and the resulting chromatin fragments were immunoprecipitated with anti-acetyl-histone H3 (Lys9), anti-dimethyl-histone H3 (Lys4), anti-trimethyl-histone H3 (Lys4), anti-dimethyl-histone H3 (Lys9), and anti-trimentyl-histone H3 (Lys36) antibodies (Millipore).

    Techniques: Chromatin Immunoprecipitation, Modification, Digital PCR

    ChIP assay to monitor interaction between H3AcK9 and PNPLA3 promoter

    Journal: Alcohol (Fayetteville, N.Y.)

    Article Title: Binge alcohol alters PNPLA3 levels in liver through epigenetic mechanism involving histone H3 acetylation

    doi: 10.1016/j.alcohol.2017.01.009

    Figure Lengend Snippet: ChIP assay to monitor interaction between H3AcK9 and PNPLA3 promoter

    Article Snippet: For complexing primary antibody and magnetic beads, 4 μg of anti-H3AcK9 (Anti-Acetyl Histone H3-lys 9, catalog # 06-942 from Millipore) or 4 μg of normal mouse IgG (Normal Mouse IgG, catalog #12-371 from Millipore) were added to 200 μL of a precleared magnetic bead slurry and incubated overnight at 4 °C with rotation.

    Techniques: Chromatin Immunoprecipitation

    ChIP assay for PNPLA3 promoter association with H3AcK9 in hepatocyte [A], mouse [B], and rat [C] liver tissues

    Journal: Alcohol (Fayetteville, N.Y.)

    Article Title: Binge alcohol alters PNPLA3 levels in liver through epigenetic mechanism involving histone H3 acetylation

    doi: 10.1016/j.alcohol.2017.01.009

    Figure Lengend Snippet: ChIP assay for PNPLA3 promoter association with H3AcK9 in hepatocyte [A], mouse [B], and rat [C] liver tissues

    Article Snippet: For complexing primary antibody and magnetic beads, 4 μg of anti-H3AcK9 (Anti-Acetyl Histone H3-lys 9, catalog # 06-942 from Millipore) or 4 μg of normal mouse IgG (Normal Mouse IgG, catalog #12-371 from Millipore) were added to 200 μL of a precleared magnetic bead slurry and incubated overnight at 4 °C with rotation.

    Techniques: Chromatin Immunoprecipitation

    Lamellipodia formation and dynamics in pancreatic tumor cells are dependent upon Dyn2 expression and phosphorylation (a) Western blot analysis of stable BxPC-3 cell lines expressing either GFP alone, Dyn2-GFP, or Dyn2Y(231,597)F-GFP. (b-d) Control BxPC-3 cells stained for cortactin (b) grow in tight clusters and appear non-motile, while stable lines expressing Dyn2-GFP (c) exhibit numerous dynamic lamellipodia. (d) In comparison, mutant Dyn2Y(231,597)F-GFP expressing cells appear loosely clustered with some motile morphology. (c-d) show GFP fluorescence of the Dyn2-GFP and Dyn2Y(231,597)F-GFP. (e) Kymographs generated from movies of the lamellipodia of BxPC-3 stable cells showing the rapid protrusion rate of lamellipodia in cells expressing WT Dyn2-GFP, relative to cells expressing GFP or Dyn2Y(231/597)F -GFP. (f) Average lamellipodia protrusion speed calculated from more than 11 cells per cell line based on collected kymographs shown in (e). (g) Western blot on lysates from BxPC-3-GFP cells treated with non-targeting siRNA (NTsi) or Dyn2 siRNA (D2si). Kymographs (i) and quantitation (i) showing reduced lamellipodia extension following Dyn2 knockdown. Kymographs (j) and quantitation (k) showing lamellipodia extension of BxPC-3-GFP cells following treatment with DMSO or dynamin inhibitors Dynasore (80 μM) or MiTMAB (20 μM). Error bars represent S.E.M. Student’s t-test was used for statistics (** represents p

    Journal: Oncogene

    Article Title: Increased Expression of the Large GTPase Dynamin 2 Potentiates Metastatic Migration and Invasion of Pancreatic Ductal Carcinoma

    doi: 10.1038/onc.2011.329

    Figure Lengend Snippet: Lamellipodia formation and dynamics in pancreatic tumor cells are dependent upon Dyn2 expression and phosphorylation (a) Western blot analysis of stable BxPC-3 cell lines expressing either GFP alone, Dyn2-GFP, or Dyn2Y(231,597)F-GFP. (b-d) Control BxPC-3 cells stained for cortactin (b) grow in tight clusters and appear non-motile, while stable lines expressing Dyn2-GFP (c) exhibit numerous dynamic lamellipodia. (d) In comparison, mutant Dyn2Y(231,597)F-GFP expressing cells appear loosely clustered with some motile morphology. (c-d) show GFP fluorescence of the Dyn2-GFP and Dyn2Y(231,597)F-GFP. (e) Kymographs generated from movies of the lamellipodia of BxPC-3 stable cells showing the rapid protrusion rate of lamellipodia in cells expressing WT Dyn2-GFP, relative to cells expressing GFP or Dyn2Y(231/597)F -GFP. (f) Average lamellipodia protrusion speed calculated from more than 11 cells per cell line based on collected kymographs shown in (e). (g) Western blot on lysates from BxPC-3-GFP cells treated with non-targeting siRNA (NTsi) or Dyn2 siRNA (D2si). Kymographs (i) and quantitation (i) showing reduced lamellipodia extension following Dyn2 knockdown. Kymographs (j) and quantitation (k) showing lamellipodia extension of BxPC-3-GFP cells following treatment with DMSO or dynamin inhibitors Dynasore (80 μM) or MiTMAB (20 μM). Error bars represent S.E.M. Student’s t-test was used for statistics (** represents p

    Article Snippet: Dynamin inhibitory compounds Dynasore and MiTMAB, from Calbiochem (Cat # 324410 and 324411), were resuspended in dimethyl sulfoxide (DMSO) to 25 mM stock concentrations and stored at −20°C until use.

    Techniques: Expressing, Western Blot, Staining, Mutagenesis, Fluorescence, Generated, Quantitation Assay

    Dynamin function is required for cell migration during wound healing Confluent monolayers of the motile pancreatic cancer cell line Panc04.03 were wounded and stimulated with 50 ng/mL EGF in the presence or absence of the indicated doses of the dynamin inhibitory compounds MiTMAB (a, b) or Dynasore (c, d), or after treatment with non-targeting (NT) or Dyn2 (D2) siRNA (e, f). Phase images of cell monolayers treated with DMSO (a,c), 20 μM MiTMAB (a), or 80 μM Dynasore (c) at 1 and 5 hrs post-EGF stimulation. Increasing the dosage of either drug resulted in a dose-dependent reduction in cell migration, contributing to slower wound closure. Similarly, depletion of Dyn2 by siRNA significantly reduced cell migration speed (e, f). Efficient knockdown of Dyn2 is shown in the inset in panel (f). The average speed of the wound edge from 3 experiments are shown. Error bars represent S.E.M. Student’s t-test was used for statistics (** represents p

    Journal: Oncogene

    Article Title: Increased Expression of the Large GTPase Dynamin 2 Potentiates Metastatic Migration and Invasion of Pancreatic Ductal Carcinoma

    doi: 10.1038/onc.2011.329

    Figure Lengend Snippet: Dynamin function is required for cell migration during wound healing Confluent monolayers of the motile pancreatic cancer cell line Panc04.03 were wounded and stimulated with 50 ng/mL EGF in the presence or absence of the indicated doses of the dynamin inhibitory compounds MiTMAB (a, b) or Dynasore (c, d), or after treatment with non-targeting (NT) or Dyn2 (D2) siRNA (e, f). Phase images of cell monolayers treated with DMSO (a,c), 20 μM MiTMAB (a), or 80 μM Dynasore (c) at 1 and 5 hrs post-EGF stimulation. Increasing the dosage of either drug resulted in a dose-dependent reduction in cell migration, contributing to slower wound closure. Similarly, depletion of Dyn2 by siRNA significantly reduced cell migration speed (e, f). Efficient knockdown of Dyn2 is shown in the inset in panel (f). The average speed of the wound edge from 3 experiments are shown. Error bars represent S.E.M. Student’s t-test was used for statistics (** represents p

    Article Snippet: Dynamin inhibitory compounds Dynasore and MiTMAB, from Calbiochem (Cat # 324410 and 324411), were resuspended in dimethyl sulfoxide (DMSO) to 25 mM stock concentrations and stored at −20°C until use.

    Techniques: Migration

    Elevated dynamin expression significantly increases individual tumor cell migration (a) Low magnification phase images of the BxPC-3 stable cell lines described in Fig 2 were acquired over 11 hrs and subsequently traced to produce migration paths. The black line depicts the migratory path of a cell over the observation period. (b) Migration tracks generated from stably transfected cells expressing GFP, (c) Dyn2-GFP and (d) Dyn2Y(231/597)F -GFP. (e) Graph depicting total migration distance of tumor cells stably overexpressing GFP, Dyn2-GFP, or Dyn2Y(231/597)F -GFP (e), Dyn2-GFP-expressing cells treated with the dynamin inhibitors MiTMAB or Dynasore at the indicated concentrations (f), or control GFP-expressing cells treated with dynamin inhibitors (g) or siRNA directed against Dyn2 (h). Compared to the other cell lines, the tumor cell line expressing Dyn2-GFP exhibited a significant increase in migration that was markedly reduced by inhibiting dynamin function. Further inhibition of endogenous Dyn2 in control cells using inhibitory compounds or Dyn2 depletion also reduced cell migration. (n=100 cells per condition, average of three experiments). Student’s t-test was used for statistics (*** represents p

    Journal: Oncogene

    Article Title: Increased Expression of the Large GTPase Dynamin 2 Potentiates Metastatic Migration and Invasion of Pancreatic Ductal Carcinoma

    doi: 10.1038/onc.2011.329

    Figure Lengend Snippet: Elevated dynamin expression significantly increases individual tumor cell migration (a) Low magnification phase images of the BxPC-3 stable cell lines described in Fig 2 were acquired over 11 hrs and subsequently traced to produce migration paths. The black line depicts the migratory path of a cell over the observation period. (b) Migration tracks generated from stably transfected cells expressing GFP, (c) Dyn2-GFP and (d) Dyn2Y(231/597)F -GFP. (e) Graph depicting total migration distance of tumor cells stably overexpressing GFP, Dyn2-GFP, or Dyn2Y(231/597)F -GFP (e), Dyn2-GFP-expressing cells treated with the dynamin inhibitors MiTMAB or Dynasore at the indicated concentrations (f), or control GFP-expressing cells treated with dynamin inhibitors (g) or siRNA directed against Dyn2 (h). Compared to the other cell lines, the tumor cell line expressing Dyn2-GFP exhibited a significant increase in migration that was markedly reduced by inhibiting dynamin function. Further inhibition of endogenous Dyn2 in control cells using inhibitory compounds or Dyn2 depletion also reduced cell migration. (n=100 cells per condition, average of three experiments). Student’s t-test was used for statistics (*** represents p

    Article Snippet: Dynamin inhibitory compounds Dynasore and MiTMAB, from Calbiochem (Cat # 324410 and 324411), were resuspended in dimethyl sulfoxide (DMSO) to 25 mM stock concentrations and stored at −20°C until use.

    Techniques: Expressing, Migration, Stable Transfection, Generated, Transfection, Inhibition

    Dyn2 function is essential for chemotactic invasion in vitro Fluorescence images of tumor cells plated on transwell filters. 2×10 5 stable BxPC-3 cells expressing GFP (a), Dyn2-GFP (b) or Dyn2Y(231/597)F -GFP (c), were plated in low serum medium (0.2%) in the top of a blind-well chamber, separated from high serum (10% FBS) in the bottom chamber by a gelatin-coated filter containing 8 μm pores and returned to the culture incubator for 4 hrs. After fixation, the cells on the filters were co-stained with phalloidin for actin (white) to visualize cell bodies and DAPI (blue) to visualize nuclei. The image focal plane in a-f represents the bottom of each filter and reveals cells that have successfully translocated by the emergence of a blue nucleus. While numerous Dyn2-GFP expressing cells have migrated through the filter (b), most cells expressing the GFP or Dyn2Y(231/597)F-GFP protein remain at the top of the chamber (a,c). These same confocal images were reoriented as brightest point projection Z-series, with the filter indicated by the yellow double lines (a’-c’). Note the marked increase in nuclei of Dyn2-GFP expressing cells below the filter compared to cells expressing Dyn2Y(231,597)F-GFP or the control. (g) Quantification of the percentage of cells that crossed a gelatin-coated filter in blind-well assays. Notably, twice as many Dyn2-GFP-expressing cells migrated across the filter as did cells expressing GFP, and more than 3 times the number compared to Dyn2Y(231/597)F-GFP. (d-f) Confocal images of the same Dyn2-GFP expressing cell line plated on filters as above and treated with DMSO as a control or the dynamin inhibitory compounds Dynasore (50 μM) and MiTMAB (10 μM). Migratory invasion through the filters was almost completely prevented in the presence of dynamin inhibitory compounds MiTMAB and Dynasore as shown clearly in the Z-series confocal images of these fields (d’-f’) and quantitated in (h). Similarly, the basal level of migration in GFP-expressing control cells was attenuated after depletion (i) or small-molecule inhibition of (j) endogenous Dyn2. Average +/− S.E.M of greater than 150 cells per line. Student’s t-test was used for statistics (**represents p

    Journal: Oncogene

    Article Title: Increased Expression of the Large GTPase Dynamin 2 Potentiates Metastatic Migration and Invasion of Pancreatic Ductal Carcinoma

    doi: 10.1038/onc.2011.329

    Figure Lengend Snippet: Dyn2 function is essential for chemotactic invasion in vitro Fluorescence images of tumor cells plated on transwell filters. 2×10 5 stable BxPC-3 cells expressing GFP (a), Dyn2-GFP (b) or Dyn2Y(231/597)F -GFP (c), were plated in low serum medium (0.2%) in the top of a blind-well chamber, separated from high serum (10% FBS) in the bottom chamber by a gelatin-coated filter containing 8 μm pores and returned to the culture incubator for 4 hrs. After fixation, the cells on the filters were co-stained with phalloidin for actin (white) to visualize cell bodies and DAPI (blue) to visualize nuclei. The image focal plane in a-f represents the bottom of each filter and reveals cells that have successfully translocated by the emergence of a blue nucleus. While numerous Dyn2-GFP expressing cells have migrated through the filter (b), most cells expressing the GFP or Dyn2Y(231/597)F-GFP protein remain at the top of the chamber (a,c). These same confocal images were reoriented as brightest point projection Z-series, with the filter indicated by the yellow double lines (a’-c’). Note the marked increase in nuclei of Dyn2-GFP expressing cells below the filter compared to cells expressing Dyn2Y(231,597)F-GFP or the control. (g) Quantification of the percentage of cells that crossed a gelatin-coated filter in blind-well assays. Notably, twice as many Dyn2-GFP-expressing cells migrated across the filter as did cells expressing GFP, and more than 3 times the number compared to Dyn2Y(231/597)F-GFP. (d-f) Confocal images of the same Dyn2-GFP expressing cell line plated on filters as above and treated with DMSO as a control or the dynamin inhibitory compounds Dynasore (50 μM) and MiTMAB (10 μM). Migratory invasion through the filters was almost completely prevented in the presence of dynamin inhibitory compounds MiTMAB and Dynasore as shown clearly in the Z-series confocal images of these fields (d’-f’) and quantitated in (h). Similarly, the basal level of migration in GFP-expressing control cells was attenuated after depletion (i) or small-molecule inhibition of (j) endogenous Dyn2. Average +/− S.E.M of greater than 150 cells per line. Student’s t-test was used for statistics (**represents p

    Article Snippet: Dynamin inhibitory compounds Dynasore and MiTMAB, from Calbiochem (Cat # 324410 and 324411), were resuspended in dimethyl sulfoxide (DMSO) to 25 mM stock concentrations and stored at −20°C until use.

    Techniques: In Vitro, Fluorescence, Expressing, Staining, Migration, Inhibition

    Chromatin immunoprecipitation using antibodies specific for methylated, unmodified and acetylated histones . Representative results of quantified DNA derived from unstimulated (basal) and 5-aza-CdR- or TSA-stimulated DU145 and MCF-7 cells immunoprecipitated by antibodies specific for methylated histones (left diagrams) as well as for unmodified and acetylated histones (right diagrams). Examples of PTEN in DU145 cells (A, B), CD44 in MCF-7 cells (C, D), GLIPR1 in DU145 cells (E, F) and Cyclin D2 in DU145 cells (G, H) are shown. All values obtained were normalized and referred to 100% of the input DNA. IgG, negative control; H3K9, Lysine 9 of histone H3; H3K4, Lysine 4 of histone H3; H4K20, Lysine 20 of histone H4; H2A, histone H2A; H2B, histone H2B; me, mono-methylated; me2, dimethylated; me3, trimethylated; Ac, acetylated.

    Journal: BMC Cancer

    Article Title: Promoter- and cell-specific epigenetic regulation of CD44, Cyclin D2, GLIPR1 and PTEN by Methyl-CpG binding proteins and histone modifications

    doi: 10.1186/1471-2407-10-297

    Figure Lengend Snippet: Chromatin immunoprecipitation using antibodies specific for methylated, unmodified and acetylated histones . Representative results of quantified DNA derived from unstimulated (basal) and 5-aza-CdR- or TSA-stimulated DU145 and MCF-7 cells immunoprecipitated by antibodies specific for methylated histones (left diagrams) as well as for unmodified and acetylated histones (right diagrams). Examples of PTEN in DU145 cells (A, B), CD44 in MCF-7 cells (C, D), GLIPR1 in DU145 cells (E, F) and Cyclin D2 in DU145 cells (G, H) are shown. All values obtained were normalized and referred to 100% of the input DNA. IgG, negative control; H3K9, Lysine 9 of histone H3; H3K4, Lysine 4 of histone H3; H4K20, Lysine 20 of histone H4; H2A, histone H2A; H2B, histone H2B; me, mono-methylated; me2, dimethylated; me3, trimethylated; Ac, acetylated.

    Article Snippet: After blocking, the membrane was probed with a 1:500 or 1:1000 dilution of antibodies which are directed against the following human proteins: MBD1, MeCP2 (Abcam, Cambridge, UK), MBD2, monomethylated lysine 9 of histone H3 (H3K9me), dimethylated lysine 9 of histone H3 (H3K9me2), trimethylated lysine 9 of histone H3 (H3K9me3), monomethylated lysine 4 of histone H3 (H3K4me), dimethylated lysine 4 of histone H3 (H3K4me2), trimethylated lysine 4 of histone H3 (H3K4me3), monomethylated lysine 20 of histone H4 (H4K20me), dimethylated lysine 20 of histone H4 (H4K20me2), trimethylated lysine 20 of histone H4 (H4K20me3) (Millipore, Schwalbach, Germany).

    Techniques: Chromatin Immunoprecipitation, Methylation, Derivative Assay, Immunoprecipitation, Negative Control