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  • 94
    Name:
    Histone H2B 53H3 Mouse mAb
    Description:
    The nucleosome made up of four core histone proteins H2A H2B H3 and H4 is the primary building block of chromatin Originally thought to function as a static scaffold for DNA packaging histones have now been shown to be dynamic proteins undergoing multiple types of post translational modifications including acetylation phosphorylation methylation and ubiquitination 1 2 The p300 CBP histone acetyltransferases acetylate multiple lysine residues in the amino terminal tail of histone H2B Lys5 12 15 and 20 at gene promoters during transcriptional activation 1 3 Hyper acetylation of the histone tails neutralizes the positive charge of these domains and is believed to weaken histone DNA and nucleosome nucleosome interactions thereby destabilizing chromatin structure and increasing the access of DNA to various DNA binding proteins 4 5 In addition acetylation of specific lysine residues creates docking sites that facilitate recruitment of many transcription and chromatin regulatory proteins that contain a bromodomain which binds to acetylated lysine residues 6 Histone H2B is mono ubiquitinated at Lys120 during transcriptional activation by the RAD6 E2 protein in conjunction with the BRE1A BRE1B E3 ligase also known as RNF20 RNF40 7 Mono ubiquitinated histone H2B Lys120 is associated with the transcribed region of active genes and stimulates transcriptional elongation by facilitating FACT dependent chromatin remodeling 7 9 In addition it is essential for subsequent methylation of histone H3 Lys4 and Lys79 two additional histone modifications that regulate transcriptional initiation and elongation 10 In response to metabolic stress AMPK is recruited to responsive genes and phosphorylates histone H2B at Lys36 both at promoters and in transcribed regions of genes and may regulate transcriptional elongation 11 In response to multiple apoptotic stimuli histone H2B is phosphorylated at Ser14 by the Mst1 kinase 12 Upon induction of apoptosis Mst1 is cleaved and activated by caspase 3 leading to global phosphorylation of histone H2B during chromatin condensation Interestingly histone H2B is rapidly phosphorylated at irradiation induced DNA damage foci in mouse embryonic fibroblasts 13 In this case phosphorylation at Ser14 is rapid depends on prior phosphorylation of H2AX Ser139 and occurs in the absence of apoptosis suggesting that Ser14 phosphorylation may have distinct roles in DNA damage repair and apoptosis
    Catalog Number:
    2934
    Price:
    None
    Applications:
    Western Blot
    Category:
    Primary Antibodies
    Source:
    Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the carboxy-terminal residues of histone H2B.
    Reactivity:
    Human Mouse Rat Monkey Zebrafish
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc h2b
    The linker and lysine-rich domain of USP42 are necessary for appropriate localization of USP42 and interaction of histone <t>H2B.</t> A , endogenous coimmunoprecipitation showing specific interaction of USP42 and H2B. B , schematic representation of the USP42 overexpression mutants used in subsequent experiments. C , immunoprecipitation of GFP-USP42 mutants to determine their interaction with H2B. Although the catalytic activity of USP42 is not required to bind H2B, deletion of the linker domain causes loss of H2B association, and deletion of the lysine-rich domain leads to a reduction of interaction. D , confocal immunofluorescence visualizing the localization of GFP-USP42 wild type and mutant proteins. Although the catalytic activity of USP42 does not alter the localization of USP42, deletion of the linker domain causes accumulation in the nucleoli, and deletion of the lysine-rich domain leads to a diffuse nuclear localization. E , in vitro deubiquitylation assay of Ubi-H2B by USP42. Wild type USP42 efficiently deubiquitylates Ubi-H2B. Disruption of the DUB domain renders USP42 inactive toward Ubi-H2B. ΔKK mutant and Δlinker mutants, which both harbor an intact DUB domain, retain the ability to deubiquitylate histone H2B. F , quantification of E . Ubi-H2B Western blots shown in E were quantified using the LI-COR Biosciences Odyssey. G, In vitro deubiquitylation assay of Ubi-H2B and Ubi-H2A by USP42. H , depletion of USP42 leads to an increase in H2B ubiquitylation and does not alter H2A ubiquitination. LI-COR quantification showing levels of H2B and H2A ubiquitylation ( Ubi-Histone ) normalized to unmodified histone with and without USP42 knockdown. Error bars represent the S.D. of three independent replicas. IP , immunoprecipitation; CTL , control.
    The nucleosome made up of four core histone proteins H2A H2B H3 and H4 is the primary building block of chromatin Originally thought to function as a static scaffold for DNA packaging histones have now been shown to be dynamic proteins undergoing multiple types of post translational modifications including acetylation phosphorylation methylation and ubiquitination 1 2 The p300 CBP histone acetyltransferases acetylate multiple lysine residues in the amino terminal tail of histone H2B Lys5 12 15 and 20 at gene promoters during transcriptional activation 1 3 Hyper acetylation of the histone tails neutralizes the positive charge of these domains and is believed to weaken histone DNA and nucleosome nucleosome interactions thereby destabilizing chromatin structure and increasing the access of DNA to various DNA binding proteins 4 5 In addition acetylation of specific lysine residues creates docking sites that facilitate recruitment of many transcription and chromatin regulatory proteins that contain a bromodomain which binds to acetylated lysine residues 6 Histone H2B is mono ubiquitinated at Lys120 during transcriptional activation by the RAD6 E2 protein in conjunction with the BRE1A BRE1B E3 ligase also known as RNF20 RNF40 7 Mono ubiquitinated histone H2B Lys120 is associated with the transcribed region of active genes and stimulates transcriptional elongation by facilitating FACT dependent chromatin remodeling 7 9 In addition it is essential for subsequent methylation of histone H3 Lys4 and Lys79 two additional histone modifications that regulate transcriptional initiation and elongation 10 In response to metabolic stress AMPK is recruited to responsive genes and phosphorylates histone H2B at Lys36 both at promoters and in transcribed regions of genes and may regulate transcriptional elongation 11 In response to multiple apoptotic stimuli histone H2B is phosphorylated at Ser14 by the Mst1 kinase 12 Upon induction of apoptosis Mst1 is cleaved and activated by caspase 3 leading to global phosphorylation of histone H2B during chromatin condensation Interestingly histone H2B is rapidly phosphorylated at irradiation induced DNA damage foci in mouse embryonic fibroblasts 13 In this case phosphorylation at Ser14 is rapid depends on prior phosphorylation of H2AX Ser139 and occurs in the absence of apoptosis suggesting that Ser14 phosphorylation may have distinct roles in DNA damage repair and apoptosis
    https://www.bioz.com/result/h2b/product/Cell Signaling Technology Inc
    Average 94 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    h2b - by Bioz Stars, 2020-09
    94/100 stars

    Images

    1) Product Images from "Ubiquitin-specific Peptidase 42 (USP42) Functions to Deubiquitylate Histones and Regulate Transcriptional Activity *"

    Article Title: Ubiquitin-specific Peptidase 42 (USP42) Functions to Deubiquitylate Histones and Regulate Transcriptional Activity *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.589267

    The linker and lysine-rich domain of USP42 are necessary for appropriate localization of USP42 and interaction of histone H2B. A , endogenous coimmunoprecipitation showing specific interaction of USP42 and H2B. B , schematic representation of the USP42 overexpression mutants used in subsequent experiments. C , immunoprecipitation of GFP-USP42 mutants to determine their interaction with H2B. Although the catalytic activity of USP42 is not required to bind H2B, deletion of the linker domain causes loss of H2B association, and deletion of the lysine-rich domain leads to a reduction of interaction. D , confocal immunofluorescence visualizing the localization of GFP-USP42 wild type and mutant proteins. Although the catalytic activity of USP42 does not alter the localization of USP42, deletion of the linker domain causes accumulation in the nucleoli, and deletion of the lysine-rich domain leads to a diffuse nuclear localization. E , in vitro deubiquitylation assay of Ubi-H2B by USP42. Wild type USP42 efficiently deubiquitylates Ubi-H2B. Disruption of the DUB domain renders USP42 inactive toward Ubi-H2B. ΔKK mutant and Δlinker mutants, which both harbor an intact DUB domain, retain the ability to deubiquitylate histone H2B. F , quantification of E . Ubi-H2B Western blots shown in E were quantified using the LI-COR Biosciences Odyssey. G, In vitro deubiquitylation assay of Ubi-H2B and Ubi-H2A by USP42. H , depletion of USP42 leads to an increase in H2B ubiquitylation and does not alter H2A ubiquitination. LI-COR quantification showing levels of H2B and H2A ubiquitylation ( Ubi-Histone ) normalized to unmodified histone with and without USP42 knockdown. Error bars represent the S.D. of three independent replicas. IP , immunoprecipitation; CTL , control.
    Figure Legend Snippet: The linker and lysine-rich domain of USP42 are necessary for appropriate localization of USP42 and interaction of histone H2B. A , endogenous coimmunoprecipitation showing specific interaction of USP42 and H2B. B , schematic representation of the USP42 overexpression mutants used in subsequent experiments. C , immunoprecipitation of GFP-USP42 mutants to determine their interaction with H2B. Although the catalytic activity of USP42 is not required to bind H2B, deletion of the linker domain causes loss of H2B association, and deletion of the lysine-rich domain leads to a reduction of interaction. D , confocal immunofluorescence visualizing the localization of GFP-USP42 wild type and mutant proteins. Although the catalytic activity of USP42 does not alter the localization of USP42, deletion of the linker domain causes accumulation in the nucleoli, and deletion of the lysine-rich domain leads to a diffuse nuclear localization. E , in vitro deubiquitylation assay of Ubi-H2B by USP42. Wild type USP42 efficiently deubiquitylates Ubi-H2B. Disruption of the DUB domain renders USP42 inactive toward Ubi-H2B. ΔKK mutant and Δlinker mutants, which both harbor an intact DUB domain, retain the ability to deubiquitylate histone H2B. F , quantification of E . Ubi-H2B Western blots shown in E were quantified using the LI-COR Biosciences Odyssey. G, In vitro deubiquitylation assay of Ubi-H2B and Ubi-H2A by USP42. H , depletion of USP42 leads to an increase in H2B ubiquitylation and does not alter H2A ubiquitination. LI-COR quantification showing levels of H2B and H2A ubiquitylation ( Ubi-Histone ) normalized to unmodified histone with and without USP42 knockdown. Error bars represent the S.D. of three independent replicas. IP , immunoprecipitation; CTL , control.

    Techniques Used: Over Expression, Immunoprecipitation, Activity Assay, Immunofluorescence, Mutagenesis, In Vitro, Western Blot, CTL Assay

    USP42 knockdown increases endogenous H2B ubiquitylation specifically on the start and early extension sites of the p21 promoter upon its induction. A–D , chromatin immunoprecipitations showing the ubiquitylation status of H2B and H2A on the p21 gene following USP42 knockdown. H2B ubiquitylation ( ubi-H2B ) increases upon p21 induction at the initiator and the first intron. This is further increased by knockdown of USP42 ( A ), whereas H2A ubiquitylation ( ubi-H2A ) is not influenced by p21 induction or USP42 knockdown ( B ). The difference in ubiquitylation is not an indirect result of general histone deposition because H2B and H2A levels are not altered ( C and D ). Error bars represent the S.D. of three independent replicas. E , expression analysis of p21 mRNA by quantitative RT-PCR. Knockdown of USP42 decreases p21 mRNA up to 38 h. F , chromatin immunoprecipitation showing the recruitment of RNA Pol II to the p21 gene upon p53 induction. USP42 knockdown ( red ) increases RNA Pol II levels closer to the start site of transcription, whereas a reduced amount of RNA Pol II is observed in the distal part of the gene relative to control ( blue ). Error bars represent the S.D. of three independent replicas. ActD , actinomycin D; CTL , control; rel. , relative; p53BS , p53 binding site.
    Figure Legend Snippet: USP42 knockdown increases endogenous H2B ubiquitylation specifically on the start and early extension sites of the p21 promoter upon its induction. A–D , chromatin immunoprecipitations showing the ubiquitylation status of H2B and H2A on the p21 gene following USP42 knockdown. H2B ubiquitylation ( ubi-H2B ) increases upon p21 induction at the initiator and the first intron. This is further increased by knockdown of USP42 ( A ), whereas H2A ubiquitylation ( ubi-H2A ) is not influenced by p21 induction or USP42 knockdown ( B ). The difference in ubiquitylation is not an indirect result of general histone deposition because H2B and H2A levels are not altered ( C and D ). Error bars represent the S.D. of three independent replicas. E , expression analysis of p21 mRNA by quantitative RT-PCR. Knockdown of USP42 decreases p21 mRNA up to 38 h. F , chromatin immunoprecipitation showing the recruitment of RNA Pol II to the p21 gene upon p53 induction. USP42 knockdown ( red ) increases RNA Pol II levels closer to the start site of transcription, whereas a reduced amount of RNA Pol II is observed in the distal part of the gene relative to control ( blue ). Error bars represent the S.D. of three independent replicas. ActD , actinomycin D; CTL , control; rel. , relative; p53BS , p53 binding site.

    Techniques Used: Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, CTL Assay, Binding Assay

    2) Product Images from "DICER- and MMSET-catalyzed H4K20me2 recruits the nucleotide excision repair factor XPA to DNA damage sites"

    Article Title: DICER- and MMSET-catalyzed H4K20me2 recruits the nucleotide excision repair factor XPA to DNA damage sites

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201704028

    MMSET enzymatic activity is required to set the H4K20me2 mark. (A) MMSET expression can restore formation of H4K20me2 foci. The graph shows the percentage of cells showing foci of H4K20me2 in unexposed cells (−UV) and 2 h after UV exposure (+UV) in shMMSET U2OS cells transfected with either mCherry, MMSET-mCherry or MMSET CDM -mCherry (mean ± SEM). Cells were counted from three independent experiments, with 200 cells counted per experiment. (B) MMSET CDM tethered to a chromatin array is unable to set the H4K20me2 mark after UV exposure. (Left) Immunofluorescence images showing H4K20me2 distribution in U2OS 2–6-3 cells, expressing mCherry-LacR-MMSET CDM (MMSET CDM -LacR). Bars, 5 µm. Arrowhead marks the position of the array. The graph on the right shows a quantification of the number of arrays showing an H4K20me2 mark in unexposed cells (−UV) and 2 h after UV exposure (+UV; mean ± SEM). Colocalization was measured from three independent experiments, with 30 cells per experiment. (C) MMSET is recruited to chromatin upon UV damage. The blot on the left shows levels of HA-MMSET in the chromatin fraction at the indicated time points after UV exposure. H2B is used as a loading control. The graph on the right shows the quantification of the relative band intensity of HA-MMSET in the indicated samples. Intensity was measured from three independent experiments.
    Figure Legend Snippet: MMSET enzymatic activity is required to set the H4K20me2 mark. (A) MMSET expression can restore formation of H4K20me2 foci. The graph shows the percentage of cells showing foci of H4K20me2 in unexposed cells (−UV) and 2 h after UV exposure (+UV) in shMMSET U2OS cells transfected with either mCherry, MMSET-mCherry or MMSET CDM -mCherry (mean ± SEM). Cells were counted from three independent experiments, with 200 cells counted per experiment. (B) MMSET CDM tethered to a chromatin array is unable to set the H4K20me2 mark after UV exposure. (Left) Immunofluorescence images showing H4K20me2 distribution in U2OS 2–6-3 cells, expressing mCherry-LacR-MMSET CDM (MMSET CDM -LacR). Bars, 5 µm. Arrowhead marks the position of the array. The graph on the right shows a quantification of the number of arrays showing an H4K20me2 mark in unexposed cells (−UV) and 2 h after UV exposure (+UV; mean ± SEM). Colocalization was measured from three independent experiments, with 30 cells per experiment. (C) MMSET is recruited to chromatin upon UV damage. The blot on the left shows levels of HA-MMSET in the chromatin fraction at the indicated time points after UV exposure. H2B is used as a loading control. The graph on the right shows the quantification of the relative band intensity of HA-MMSET in the indicated samples. Intensity was measured from three independent experiments.

    Techniques Used: Activity Assay, Expressing, Transfection, Immunofluorescence

    MMSET mediated H4K20me2 is required for recruitment of XPA. (A) MMSET-mediated H4K20me2 recruits XPA to chromatin. Immunofluorescence images showing distribution of XPA-EGFP and H4K20me2 in cells with MMSET-LacR tethered arrays in both UV-unexposed and exposed cells (left). The graph on the right shows a quantification of the number of arrays showing XPA-EGFP recruitment in unexposed cells (−UV) and 2 h after UV exposure (+UV; mean ± SEM). Colocalization was measured from three independent experiments, with 30 cells per experiment. Bars, 5 µm. Statistical significance was assessed by an unpaired t test. **, P ≤ 0.001. (B) DICER can also recruit XPA to chromatin. Immunofluorescence images showing distribution of XPA-EGFP in cells with DICER-LacR tethered arrays, in both UV unexposed and exposed cells. Bars, 5 µm. (C) MMSET is required for DICER mediated XPA recruitment. The graph shows a quantification of the number of DICER-LacR arrays showing XPA-EGFP recruitment in the indicated knockdown cell lines (mean ± SEM). Colocalization was measured from three independent experiments, with 30 cells per experiment. Statistical significance was assessed by an unpaired t test. ***, P ≤ 0.0001. (D) MMSET is required for XPA recruitment to localized sites of DNA damage. Immunofluorescence images showing distribution of CPDs and XPA in U2OS cells subjected to damage through a 3-µm micropore membrane. XPA recruitment to CPDs was quantified in both control (shNMC) and MMSET knockdown (shMMSET) cell lines. Bar, 10 µm. (E) MMSET is required for XPA recruitment to chromatin. Western blot showing chromatin association experiment in unexposed cells (−UV) and cells after 2 h UV exposure (+UV) in the indicated knockdown cell lines (right). Quantification of the relative intensity of XPA in the indicated conditions (left). H2B was used as a loading control (mean± SEM; n = 3).
    Figure Legend Snippet: MMSET mediated H4K20me2 is required for recruitment of XPA. (A) MMSET-mediated H4K20me2 recruits XPA to chromatin. Immunofluorescence images showing distribution of XPA-EGFP and H4K20me2 in cells with MMSET-LacR tethered arrays in both UV-unexposed and exposed cells (left). The graph on the right shows a quantification of the number of arrays showing XPA-EGFP recruitment in unexposed cells (−UV) and 2 h after UV exposure (+UV; mean ± SEM). Colocalization was measured from three independent experiments, with 30 cells per experiment. Bars, 5 µm. Statistical significance was assessed by an unpaired t test. **, P ≤ 0.001. (B) DICER can also recruit XPA to chromatin. Immunofluorescence images showing distribution of XPA-EGFP in cells with DICER-LacR tethered arrays, in both UV unexposed and exposed cells. Bars, 5 µm. (C) MMSET is required for DICER mediated XPA recruitment. The graph shows a quantification of the number of DICER-LacR arrays showing XPA-EGFP recruitment in the indicated knockdown cell lines (mean ± SEM). Colocalization was measured from three independent experiments, with 30 cells per experiment. Statistical significance was assessed by an unpaired t test. ***, P ≤ 0.0001. (D) MMSET is required for XPA recruitment to localized sites of DNA damage. Immunofluorescence images showing distribution of CPDs and XPA in U2OS cells subjected to damage through a 3-µm micropore membrane. XPA recruitment to CPDs was quantified in both control (shNMC) and MMSET knockdown (shMMSET) cell lines. Bar, 10 µm. (E) MMSET is required for XPA recruitment to chromatin. Western blot showing chromatin association experiment in unexposed cells (−UV) and cells after 2 h UV exposure (+UV) in the indicated knockdown cell lines (right). Quantification of the relative intensity of XPA in the indicated conditions (left). H2B was used as a loading control (mean± SEM; n = 3).

    Techniques Used: Immunofluorescence, Western Blot

    3) Product Images from "The contribution of N2O3 to the cytotoxicity of the nitric oxide donor DETA/NO: an emerging role for S-nitrosylation"

    Article Title: The contribution of N2O3 to the cytotoxicity of the nitric oxide donor DETA/NO: an emerging role for S-nitrosylation

    Journal: Bioscience Reports

    doi: 10.1042/BSR20120120

    Biotin switch assay to determine the extent of protein S-nitrosylation following exposure to NO • and ONOO − RSNO profile in MDA-MB-231 cells following the treatment with ONOO − (1 mM), AS (1 mM), or LD 50 concentrations of DETA/NO under normoxia or 0.1% hypoxia ( * ), (2) or MDA-MB-231 cells treated with DETA/NO±sodium azide. (3) The S-nitrosylated total protein profile in nuclear extracts following the treatment with LD 50 concentrations of DETA/NO. Whole cell or nuclear lysates were subjected to the biotin switch method, biotin labelled nitrosoproteins were separated on SDS/PAGE (10% gel) and detected by Western blot analysis with an anti-biotin antibody. In parallel, β-actin or H2B protein levels served as a loading control. Images shown are the representative blots of three independent experiments. Arrows on the left point to visible bands representing the molecular mass of S-nitrosylated proteins.
    Figure Legend Snippet: Biotin switch assay to determine the extent of protein S-nitrosylation following exposure to NO • and ONOO − RSNO profile in MDA-MB-231 cells following the treatment with ONOO − (1 mM), AS (1 mM), or LD 50 concentrations of DETA/NO under normoxia or 0.1% hypoxia ( * ), (2) or MDA-MB-231 cells treated with DETA/NO±sodium azide. (3) The S-nitrosylated total protein profile in nuclear extracts following the treatment with LD 50 concentrations of DETA/NO. Whole cell or nuclear lysates were subjected to the biotin switch method, biotin labelled nitrosoproteins were separated on SDS/PAGE (10% gel) and detected by Western blot analysis with an anti-biotin antibody. In parallel, β-actin or H2B protein levels served as a loading control. Images shown are the representative blots of three independent experiments. Arrows on the left point to visible bands representing the molecular mass of S-nitrosylated proteins.

    Techniques Used: Biotin Switch Assay, Multiple Displacement Amplification, SDS Page, Western Blot

    4) 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

    5) Product Images from "TRIM37 is a new histone H2A ubiquitin ligase and breast cancer oncoprotein"

    Article Title: TRIM37 is a new histone H2A ubiquitin ligase and breast cancer oncoprotein

    Journal: Nature

    doi: 10.1038/nature13955

    Control experiments for Fig. 1 a , ChIP monitoring H2A-ub enrichment on Fas in Kras NIH 3T3 cells expressing a non-silencing (NS) shRNA or a Trim37, Rnf2 or Bmi1 shRNA. Three regions of the Fas promoter were analysed: the core promoter/transcription start site (CP/TSS), and 1 and 2 kb upstream of the TSS. Actb and Gapdh are shown as negative controls. Error bars indicate SEM; n=9 (three biological replicates with three technical replicates per sample). b , H2A-ub ChIP as described in (a) using a second Trim37, Rnf2 or Bmi1 shRNA unrelated to that used in (a), c , qRT-PCR analysis monitoring knockdown efficiencies of Trim37 and Rnf2 shRNAs in NIH 3T3 cells. The results are given relative to expression following treatment with a NS shRNA, which was set to 1. For knockdown efficiencies of the Bmi1 shRNAs see Gazin et al., 2007 (ref. 6 ). Error bars indicate SEM; n=3 technical replicates of a representative experiment (out of three experiments). d , In vitro ubiquitination assay. Purified H2B was incubated with E1 (UBE1), E2 (UBCH5B), E3 (TRIM37 or BRCA1), ATP and HA-ub. Blots were probed with antibodies against H2B-ub or H2B. The results show that TRIM37 does not ubiquitinate H2B. BRCA1, which is known to ubiquitinate H2B at K120 39 , 40 , was used as a positive control. e, f , qRT-PCR (e) and immunoblots (f) monitoring TRIM37 and RNF2 in various cell lines. Expression of TRIM37 and RNF2 expression were was normalized to that obtained in HMECs, which was set to 1. Error bars indicate SD; n=3 technical replicates of a representative experiment (out of three experiments). g , Immunoblot monitoring TRIM37 levels in MCF7 cells expressing a NS or one of two unrelated TRIM37 shRNAs. α-tubulin (TUBA) was monitored as a loading control. The results indicate that the TRIM37 antibody is highly specific. h , qRT-PCR analysis monitoring knockdown efficiencies of TRIM37 shRNAs in MCF7, BT474 and MDA-MD-361 cells. Error bars indicate SEM; n=3 technical replicates of a representative experiment (out of three experiments). i , (Top) Immunoblots monitoring levels of H2A-ub and H2B-ub in MCF7 cells expressing a NS or one of two unrelated TRIM37 shRNAs. (Bottom) Quantification of the H2A-ub immunoblots relative to TUBA. The relative level of H2A-ub in NS cells was set to 1. In this experiment, histones were acid extracted. j , (Top) Immunoblots monitoring TRIM37, H2A-ub, H2A, H2B-ub and H4 in BT474 and MDA-MB-361 cells expressing a NS or one of two unrelated TRIM37 shRNAs. (Bottom) H2A-ub quantification, as described in (g). k , qRT-PCR analysis monitoring knockdown efficiencies of RNF2 shRNAs in MCF7 cells. Error bars indicate SEM; n=3 technical replicates of a representative experiment (out of three experiments). l , (Top) Immunoblots monitoring TRIM37, H2A-ub, H2A and H4 in MDA-MB-231, Hs578T and T47D cells expressing a NS or one of two unrelated TRIM37 shRNAs. (Bottom) H2A-ub quantification, as described in (g). m , Proliferation of cultured MDA-MB-231, Hs578T and T47D cells expressing a NS or TRIM37 shRNA. Error bars indicate SD; n=3 technical replicates of a representative experiment (out of three experiments). The results show that knockdown of TRIM37 has no effect on proliferation of breast cancer cell lines lacking 17q23 amplification. n , qRT-PCR analysis monitoring TRIM37 expression in an MCF10A cell line ectopically expressing TRIM37 or, as a control, empty vector. The results were normalized to TRIM37 expression in MCF10A cells expressing empty vector, which was set to 1. Error bars indicate SEM; n=3 technical replicates of a representative experiment (out of three experiments). * P
    Figure Legend Snippet: Control experiments for Fig. 1 a , ChIP monitoring H2A-ub enrichment on Fas in Kras NIH 3T3 cells expressing a non-silencing (NS) shRNA or a Trim37, Rnf2 or Bmi1 shRNA. Three regions of the Fas promoter were analysed: the core promoter/transcription start site (CP/TSS), and 1 and 2 kb upstream of the TSS. Actb and Gapdh are shown as negative controls. Error bars indicate SEM; n=9 (three biological replicates with three technical replicates per sample). b , H2A-ub ChIP as described in (a) using a second Trim37, Rnf2 or Bmi1 shRNA unrelated to that used in (a), c , qRT-PCR analysis monitoring knockdown efficiencies of Trim37 and Rnf2 shRNAs in NIH 3T3 cells. The results are given relative to expression following treatment with a NS shRNA, which was set to 1. For knockdown efficiencies of the Bmi1 shRNAs see Gazin et al., 2007 (ref. 6 ). Error bars indicate SEM; n=3 technical replicates of a representative experiment (out of three experiments). d , In vitro ubiquitination assay. Purified H2B was incubated with E1 (UBE1), E2 (UBCH5B), E3 (TRIM37 or BRCA1), ATP and HA-ub. Blots were probed with antibodies against H2B-ub or H2B. The results show that TRIM37 does not ubiquitinate H2B. BRCA1, which is known to ubiquitinate H2B at K120 39 , 40 , was used as a positive control. e, f , qRT-PCR (e) and immunoblots (f) monitoring TRIM37 and RNF2 in various cell lines. Expression of TRIM37 and RNF2 expression were was normalized to that obtained in HMECs, which was set to 1. Error bars indicate SD; n=3 technical replicates of a representative experiment (out of three experiments). g , Immunoblot monitoring TRIM37 levels in MCF7 cells expressing a NS or one of two unrelated TRIM37 shRNAs. α-tubulin (TUBA) was monitored as a loading control. The results indicate that the TRIM37 antibody is highly specific. h , qRT-PCR analysis monitoring knockdown efficiencies of TRIM37 shRNAs in MCF7, BT474 and MDA-MD-361 cells. Error bars indicate SEM; n=3 technical replicates of a representative experiment (out of three experiments). i , (Top) Immunoblots monitoring levels of H2A-ub and H2B-ub in MCF7 cells expressing a NS or one of two unrelated TRIM37 shRNAs. (Bottom) Quantification of the H2A-ub immunoblots relative to TUBA. The relative level of H2A-ub in NS cells was set to 1. In this experiment, histones were acid extracted. j , (Top) Immunoblots monitoring TRIM37, H2A-ub, H2A, H2B-ub and H4 in BT474 and MDA-MB-361 cells expressing a NS or one of two unrelated TRIM37 shRNAs. (Bottom) H2A-ub quantification, as described in (g). k , qRT-PCR analysis monitoring knockdown efficiencies of RNF2 shRNAs in MCF7 cells. Error bars indicate SEM; n=3 technical replicates of a representative experiment (out of three experiments). l , (Top) Immunoblots monitoring TRIM37, H2A-ub, H2A and H4 in MDA-MB-231, Hs578T and T47D cells expressing a NS or one of two unrelated TRIM37 shRNAs. (Bottom) H2A-ub quantification, as described in (g). m , Proliferation of cultured MDA-MB-231, Hs578T and T47D cells expressing a NS or TRIM37 shRNA. Error bars indicate SD; n=3 technical replicates of a representative experiment (out of three experiments). The results show that knockdown of TRIM37 has no effect on proliferation of breast cancer cell lines lacking 17q23 amplification. n , qRT-PCR analysis monitoring TRIM37 expression in an MCF10A cell line ectopically expressing TRIM37 or, as a control, empty vector. The results were normalized to TRIM37 expression in MCF10A cells expressing empty vector, which was set to 1. Error bars indicate SEM; n=3 technical replicates of a representative experiment (out of three experiments). * P

    Techniques Used: Chromatin Immunoprecipitation, Expressing, shRNA, Quantitative RT-PCR, In Vitro, Ubiquitin Assay, Purification, Incubation, Positive Control, Western Blot, Multiple Displacement Amplification, Cell Culture, Amplification, Plasmid Preparation

    6) Product Images from "Nuclear TBLR1 as an ER corepressor promotes cell proliferation, migration and invasion in breast and ovarian cancer"

    Article Title: Nuclear TBLR1 as an ER corepressor promotes cell proliferation, migration and invasion in breast and ovarian cancer

    Journal: American Journal of Cancer Research

    doi:

    Expression of TBLR1 in breast and ovarian cancer. (A) Localization of TBLR1 expression in breast cells was examined by cell fractionation followed by western blot. GAPDH is used as a cytoplasm loading control. H2B is used as a nuclear loading control.
    Figure Legend Snippet: Expression of TBLR1 in breast and ovarian cancer. (A) Localization of TBLR1 expression in breast cells was examined by cell fractionation followed by western blot. GAPDH is used as a cytoplasm loading control. H2B is used as a nuclear loading control.

    Techniques Used: Expressing, Cell Fractionation, Western Blot

    7) Product Images from "Regulation of B cell homeostasis and activation by the tumor suppressor gene CYLD"

    Article Title: Regulation of B cell homeostasis and activation by the tumor suppressor gene CYLD

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20070318

    Analysis of signal transduction of CYLD ex7/8 mutant splenic B cells. (A) Whole-cell extracts were examined by Western blot using antibodies against the indicated proteins. Shown are three Western blots from B cell lysates of three different WT (lanes 1–3) and three different CYLD ex7/8 (lanes 4–6) mice isolated in the same experiment. AKT-specific antibody was used as loading control. (B; left) cytoplasmic and (right) nuclear extracts of nonactivated B cells isolated from the indicated genotypes were probed with RelB-specific antibody. H2B and actin-specific antibodies were used as loading control. (C) MACS-purified B cells were rested for 4 h and subsequently stimulated with optimal concentrations of BAFF for the indicated time points and subjected to Western blot analysis using the indicated antibody. (D) Extracts from CYLD ex7/8 and WT B cells treated with an anti-CD40 antibody, LPS, or anti-BCR for different time points were analyzed with antibodies against IκBα and pIκBα. Actin-specific antibody was used as loading control. (E) EMSA was performed with extracts of B cells rested for 4 h after isolation. Cells were then activated by anti-BCR for the indicated time points before extracts were prepared, incubated with NF-κB–specific labeled probe, and separated on native PAGE. (F) B cell lysates from CYLD ex7/8 and WT controls were stimulated with LPS for the indicated time points, and CBA was performed using beads loaded with antibodies specific for p-p38 and pERK1/2. (G) Western blot was performed for p-p38 and pERK from B cells of the indicated genotypes stimulated with anti-BCR for the indicated time points. Actin-specific antibody was used as loading control.
    Figure Legend Snippet: Analysis of signal transduction of CYLD ex7/8 mutant splenic B cells. (A) Whole-cell extracts were examined by Western blot using antibodies against the indicated proteins. Shown are three Western blots from B cell lysates of three different WT (lanes 1–3) and three different CYLD ex7/8 (lanes 4–6) mice isolated in the same experiment. AKT-specific antibody was used as loading control. (B; left) cytoplasmic and (right) nuclear extracts of nonactivated B cells isolated from the indicated genotypes were probed with RelB-specific antibody. H2B and actin-specific antibodies were used as loading control. (C) MACS-purified B cells were rested for 4 h and subsequently stimulated with optimal concentrations of BAFF for the indicated time points and subjected to Western blot analysis using the indicated antibody. (D) Extracts from CYLD ex7/8 and WT B cells treated with an anti-CD40 antibody, LPS, or anti-BCR for different time points were analyzed with antibodies against IκBα and pIκBα. Actin-specific antibody was used as loading control. (E) EMSA was performed with extracts of B cells rested for 4 h after isolation. Cells were then activated by anti-BCR for the indicated time points before extracts were prepared, incubated with NF-κB–specific labeled probe, and separated on native PAGE. (F) B cell lysates from CYLD ex7/8 and WT controls were stimulated with LPS for the indicated time points, and CBA was performed using beads loaded with antibodies specific for p-p38 and pERK1/2. (G) Western blot was performed for p-p38 and pERK from B cells of the indicated genotypes stimulated with anti-BCR for the indicated time points. Actin-specific antibody was used as loading control.

    Techniques Used: Transduction, Mutagenesis, Western Blot, Mouse Assay, Isolation, Magnetic Cell Separation, Purification, Incubation, Labeling, Clear Native PAGE, Crocin Bleaching Assay

    8) Product Images from "DICER- and MMSET-catalyzed H4K20me2 recruits the nucleotide excision repair factor XPA to DNA damage sites"

    Article Title: DICER- and MMSET-catalyzed H4K20me2 recruits the nucleotide excision repair factor XPA to DNA damage sites

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201704028

    MMSET enzymatic activity is required to set the H4K20me2 mark. (A) MMSET expression can restore formation of H4K20me2 foci. The graph shows the percentage of cells showing foci of H4K20me2 in unexposed cells (−UV) and 2 h after UV exposure (+UV) in shMMSET U2OS cells transfected with either mCherry, MMSET-mCherry or MMSET CDM -mCherry (mean ± SEM). Cells were counted from three independent experiments, with 200 cells counted per experiment. (B) MMSET CDM tethered to a chromatin array is unable to set the H4K20me2 mark after UV exposure. (Left) Immunofluorescence images showing H4K20me2 distribution in U2OS 2–6-3 cells, expressing mCherry-LacR-MMSET CDM (MMSET CDM -LacR). Bars, 5 µm. Arrowhead marks the position of the array. The graph on the right shows a quantification of the number of arrays showing an H4K20me2 mark in unexposed cells (−UV) and 2 h after UV exposure (+UV; mean ± SEM). Colocalization was measured from three independent experiments, with 30 cells per experiment. (C) MMSET is recruited to chromatin upon UV damage. The blot on the left shows levels of HA-MMSET in the chromatin fraction at the indicated time points after UV exposure. H2B is used as a loading control. The graph on the right shows the quantification of the relative band intensity of HA-MMSET in the indicated samples. Intensity was measured from three independent experiments.
    Figure Legend Snippet: MMSET enzymatic activity is required to set the H4K20me2 mark. (A) MMSET expression can restore formation of H4K20me2 foci. The graph shows the percentage of cells showing foci of H4K20me2 in unexposed cells (−UV) and 2 h after UV exposure (+UV) in shMMSET U2OS cells transfected with either mCherry, MMSET-mCherry or MMSET CDM -mCherry (mean ± SEM). Cells were counted from three independent experiments, with 200 cells counted per experiment. (B) MMSET CDM tethered to a chromatin array is unable to set the H4K20me2 mark after UV exposure. (Left) Immunofluorescence images showing H4K20me2 distribution in U2OS 2–6-3 cells, expressing mCherry-LacR-MMSET CDM (MMSET CDM -LacR). Bars, 5 µm. Arrowhead marks the position of the array. The graph on the right shows a quantification of the number of arrays showing an H4K20me2 mark in unexposed cells (−UV) and 2 h after UV exposure (+UV; mean ± SEM). Colocalization was measured from three independent experiments, with 30 cells per experiment. (C) MMSET is recruited to chromatin upon UV damage. The blot on the left shows levels of HA-MMSET in the chromatin fraction at the indicated time points after UV exposure. H2B is used as a loading control. The graph on the right shows the quantification of the relative band intensity of HA-MMSET in the indicated samples. Intensity was measured from three independent experiments.

    Techniques Used: Activity Assay, Expressing, Transfection, Immunofluorescence

    MMSET mediated H4K20me2 is required for recruitment of XPA. (A) MMSET-mediated H4K20me2 recruits XPA to chromatin. Immunofluorescence images showing distribution of XPA-EGFP and H4K20me2 in cells with MMSET-LacR tethered arrays in both UV-unexposed and exposed cells (left). The graph on the right shows a quantification of the number of arrays showing XPA-EGFP recruitment in unexposed cells (−UV) and 2 h after UV exposure (+UV; mean ± SEM). Colocalization was measured from three independent experiments, with 30 cells per experiment. Bars, 5 µm. Statistical significance was assessed by an unpaired t test. **, P ≤ 0.001. (B) DICER can also recruit XPA to chromatin. Immunofluorescence images showing distribution of XPA-EGFP in cells with DICER-LacR tethered arrays, in both UV unexposed and exposed cells. Bars, 5 µm. (C) MMSET is required for DICER mediated XPA recruitment. The graph shows a quantification of the number of DICER-LacR arrays showing XPA-EGFP recruitment in the indicated knockdown cell lines (mean ± SEM). Colocalization was measured from three independent experiments, with 30 cells per experiment. Statistical significance was assessed by an unpaired t test. ***, P ≤ 0.0001. (D) MMSET is required for XPA recruitment to localized sites of DNA damage. Immunofluorescence images showing distribution of CPDs and XPA in U2OS cells subjected to damage through a 3-µm micropore membrane. XPA recruitment to CPDs was quantified in both control (shNMC) and MMSET knockdown (shMMSET) cell lines. Bar, 10 µm. (E) MMSET is required for XPA recruitment to chromatin. Western blot showing chromatin association experiment in unexposed cells (−UV) and cells after 2 h UV exposure (+UV) in the indicated knockdown cell lines (right). Quantification of the relative intensity of XPA in the indicated conditions (left). H2B was used as a loading control (mean± SEM; n = 3).
    Figure Legend Snippet: MMSET mediated H4K20me2 is required for recruitment of XPA. (A) MMSET-mediated H4K20me2 recruits XPA to chromatin. Immunofluorescence images showing distribution of XPA-EGFP and H4K20me2 in cells with MMSET-LacR tethered arrays in both UV-unexposed and exposed cells (left). The graph on the right shows a quantification of the number of arrays showing XPA-EGFP recruitment in unexposed cells (−UV) and 2 h after UV exposure (+UV; mean ± SEM). Colocalization was measured from three independent experiments, with 30 cells per experiment. Bars, 5 µm. Statistical significance was assessed by an unpaired t test. **, P ≤ 0.001. (B) DICER can also recruit XPA to chromatin. Immunofluorescence images showing distribution of XPA-EGFP in cells with DICER-LacR tethered arrays, in both UV unexposed and exposed cells. Bars, 5 µm. (C) MMSET is required for DICER mediated XPA recruitment. The graph shows a quantification of the number of DICER-LacR arrays showing XPA-EGFP recruitment in the indicated knockdown cell lines (mean ± SEM). Colocalization was measured from three independent experiments, with 30 cells per experiment. Statistical significance was assessed by an unpaired t test. ***, P ≤ 0.0001. (D) MMSET is required for XPA recruitment to localized sites of DNA damage. Immunofluorescence images showing distribution of CPDs and XPA in U2OS cells subjected to damage through a 3-µm micropore membrane. XPA recruitment to CPDs was quantified in both control (shNMC) and MMSET knockdown (shMMSET) cell lines. Bar, 10 µm. (E) MMSET is required for XPA recruitment to chromatin. Western blot showing chromatin association experiment in unexposed cells (−UV) and cells after 2 h UV exposure (+UV) in the indicated knockdown cell lines (right). Quantification of the relative intensity of XPA in the indicated conditions (left). H2B was used as a loading control (mean± SEM; n = 3).

    Techniques Used: Immunofluorescence, Western Blot

    9) Product Images from "Regulation of Greatwall kinase by protein stabilization and nuclear localization"

    Article Title: Regulation of Greatwall kinase by protein stabilization and nuclear localization

    Journal: Cell Cycle

    doi: 10.4161/15384101.2014.962942

    Hsp90 is required for the stabilization of Gwl. ( A ) Xenopus egg extracts were prepared as described in Materials and Methods, and treated with or without Geldanamycin (2 μM). Extracts at indicated time points were analyzed by immunoblotting for Gwl, Cdc27, phospho-Cdk substrates, and H2B. ( B ) GST-Gwl was added into Xenopus egg extracts in either interphase or M-phase, and incubated overtime with Geldanamycin. Extract samples were analyzed by immunoblotting for GST.
    Figure Legend Snippet: Hsp90 is required for the stabilization of Gwl. ( A ) Xenopus egg extracts were prepared as described in Materials and Methods, and treated with or without Geldanamycin (2 μM). Extracts at indicated time points were analyzed by immunoblotting for Gwl, Cdc27, phospho-Cdk substrates, and H2B. ( B ) GST-Gwl was added into Xenopus egg extracts in either interphase or M-phase, and incubated overtime with Geldanamycin. Extract samples were analyzed by immunoblotting for GST.

    Techniques Used: Incubation

    10) Product Images from "A genome-wide IR-induced RAD51 foci RNAi screen identifies CDC73 involved in chromatin remodeling for DNA repair"

    Article Title: A genome-wide IR-induced RAD51 foci RNAi screen identifies CDC73 involved in chromatin remodeling for DNA repair

    Journal: Cell Discovery

    doi: 10.1038/celldisc.2015.34

    CDC73 silencing leads to impaired H2B ubiquitination and reduced DNA damage-induced H2B release. ( a ) CDC73 binds to Histones H2B and H3 in co-immunoprecipitation experiments. PAF1 serves as a positive control. ( b ) H2BK120Ub is reduced in CDC73 knockdown cells. The effect is enhanced after exposure to HU (2 m m ). Calculated ratios (H2Bub/H2B) are indicated below the blot. ( c ) CDC73 silencing leads to decreased ubiquitination on H2BK120, which is rescued by expression of a siRNA-resistant CDC73 construct (R) (EV, empty vector control transfection). ( d ) Low energy laser irradiation (405nm) leads to DNA damage-induced recruitment of red fluorescent protein–XRCC1. ( e ) Cells were analyzed for the migration speed of H2B–GFP away from the site of DNA damage. Quantification was done using the plot profile tool in ImageJ (inlet) in the indicated area (red square in d ) and the distance was measured at a defined gray value for start and end time points (red line). Each experiment was repeated at least 3 times and at least 10 cells were analyzed per experiment (Average and s.d. P -values are calculated with Student’s t -test and scale bar represents 5 μm). CDC73 silencing reduces the speed of H2B migration significantly (* P =0.017) and to a similar speed as the H2BK120A–GFP mutant (** P =0.006). ( f ) Representative images for the quantification in e ( t irr, timepoint of irradiation; t 120s , 2 min after irradiation). Images are displayed in pseudocolors for better illustration. ( g ) H2B and H3 bind tighter to chromatin in absence of CDC73 at a salt concentration of 0.6 m m NaCl. GAPDH and Ku70/86 are shown as soluble controls. Ratios for CDC73 knockdown and H2B and H3 in the soluble fraction are quantified and summarized in the table.
    Figure Legend Snippet: CDC73 silencing leads to impaired H2B ubiquitination and reduced DNA damage-induced H2B release. ( a ) CDC73 binds to Histones H2B and H3 in co-immunoprecipitation experiments. PAF1 serves as a positive control. ( b ) H2BK120Ub is reduced in CDC73 knockdown cells. The effect is enhanced after exposure to HU (2 m m ). Calculated ratios (H2Bub/H2B) are indicated below the blot. ( c ) CDC73 silencing leads to decreased ubiquitination on H2BK120, which is rescued by expression of a siRNA-resistant CDC73 construct (R) (EV, empty vector control transfection). ( d ) Low energy laser irradiation (405nm) leads to DNA damage-induced recruitment of red fluorescent protein–XRCC1. ( e ) Cells were analyzed for the migration speed of H2B–GFP away from the site of DNA damage. Quantification was done using the plot profile tool in ImageJ (inlet) in the indicated area (red square in d ) and the distance was measured at a defined gray value for start and end time points (red line). Each experiment was repeated at least 3 times and at least 10 cells were analyzed per experiment (Average and s.d. P -values are calculated with Student’s t -test and scale bar represents 5 μm). CDC73 silencing reduces the speed of H2B migration significantly (* P =0.017) and to a similar speed as the H2BK120A–GFP mutant (** P =0.006). ( f ) Representative images for the quantification in e ( t irr, timepoint of irradiation; t 120s , 2 min after irradiation). Images are displayed in pseudocolors for better illustration. ( g ) H2B and H3 bind tighter to chromatin in absence of CDC73 at a salt concentration of 0.6 m m NaCl. GAPDH and Ku70/86 are shown as soluble controls. Ratios for CDC73 knockdown and H2B and H3 in the soluble fraction are quantified and summarized in the table.

    Techniques Used: Immunoprecipitation, Positive Control, Expressing, Construct, Plasmid Preparation, Transfection, Irradiation, Migration, Mutagenesis, Concentration Assay

    CDC73 interacts with a number of chromatin-remodeling factors. ( a ) Co-IP with CDC73 confirmed its interaction with UBE1A, CAND1, CUL1, FBXO21, KU86, RUVBL2 and RNF20. PAF1 serves as a positive control and actin as a negative control. ( b ) The interactions are unchanged on IR treatment (2 Gy). ( c ) siRNA-mediated silencing of FBXO21, RUVBL2, CAND1, CUL1 and PAF1 leads to a decrease in H2BK120Ub. Ratios H2BK120Ub:H2B are indicated. ( d ) siRNA knockdown of CAND1, CUL1 and RUVBL2 leads to a similar decrease in homologous recombination repair as CDC73 knockdown (s.d. of average; more than three repeats). ( e ) Model describing the mode of action of CDC73 at DSB. CDC73 recruits chromatin-remodeling components that mediate H2BK120Ub and subsequent eviction of H2B from chromatin. The decondensed state of the chromatin allows repair enzymes to engage with DNA and proceed with resection and downstream DNA repair.
    Figure Legend Snippet: CDC73 interacts with a number of chromatin-remodeling factors. ( a ) Co-IP with CDC73 confirmed its interaction with UBE1A, CAND1, CUL1, FBXO21, KU86, RUVBL2 and RNF20. PAF1 serves as a positive control and actin as a negative control. ( b ) The interactions are unchanged on IR treatment (2 Gy). ( c ) siRNA-mediated silencing of FBXO21, RUVBL2, CAND1, CUL1 and PAF1 leads to a decrease in H2BK120Ub. Ratios H2BK120Ub:H2B are indicated. ( d ) siRNA knockdown of CAND1, CUL1 and RUVBL2 leads to a similar decrease in homologous recombination repair as CDC73 knockdown (s.d. of average; more than three repeats). ( e ) Model describing the mode of action of CDC73 at DSB. CDC73 recruits chromatin-remodeling components that mediate H2BK120Ub and subsequent eviction of H2B from chromatin. The decondensed state of the chromatin allows repair enzymes to engage with DNA and proceed with resection and downstream DNA repair.

    Techniques Used: Co-Immunoprecipitation Assay, Positive Control, Negative Control, Homologous Recombination

    The role of CDC73 in HRR is independent of its transcriptional function. ( a ) Illustration of the siRNA-resistant CDC73, the core mutant (ΔC) and the 227X mutant. ( b ) Immunofluorescence of the eGFP-tagged constructs in U2OS cells in combination with DAPI (blue) and Phalloidin (red). ( c ) The defect in HRR as measured by the DR–GFP assay can be rescued by re-expression of a WT (* P =0.029) and the ΔC mutant. ( d ) PAF1 silencing does reduce HRR (* P =0.009). ( e ) DRB treatment (50 μm, 2 h) has the same effect on H2B mobility as CDC73 silencing (* P =0.033). ( f ) The CDC73-227X mutant does not rescue HRR in the DR–GFP assay (** P =8×10 -4 ). ( g ) CDC73 knockdown does not affect the expression of a number of essential genes for DNA repair and has no effect on the recruitment of BRCA1 ( h ). For all experiments ( n ⩾2). Average and s.d. are plotted. P -values are calculated with Student’s t -test and scale bars represent 50 and 10 μm.
    Figure Legend Snippet: The role of CDC73 in HRR is independent of its transcriptional function. ( a ) Illustration of the siRNA-resistant CDC73, the core mutant (ΔC) and the 227X mutant. ( b ) Immunofluorescence of the eGFP-tagged constructs in U2OS cells in combination with DAPI (blue) and Phalloidin (red). ( c ) The defect in HRR as measured by the DR–GFP assay can be rescued by re-expression of a WT (* P =0.029) and the ΔC mutant. ( d ) PAF1 silencing does reduce HRR (* P =0.009). ( e ) DRB treatment (50 μm, 2 h) has the same effect on H2B mobility as CDC73 silencing (* P =0.033). ( f ) The CDC73-227X mutant does not rescue HRR in the DR–GFP assay (** P =8×10 -4 ). ( g ) CDC73 knockdown does not affect the expression of a number of essential genes for DNA repair and has no effect on the recruitment of BRCA1 ( h ). For all experiments ( n ⩾2). Average and s.d. are plotted. P -values are calculated with Student’s t -test and scale bars represent 50 and 10 μm.

    Techniques Used: Mutagenesis, Immunofluorescence, Construct, Expressing

    11) 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

    12) Product Images from "Loss of GCN5 leads to increased neuronal apoptosis by upregulating E2F1- and Egr-1-dependent BH3-only protein Bim"

    Article Title: Loss of GCN5 leads to increased neuronal apoptosis by upregulating E2F1- and Egr-1-dependent BH3-only protein Bim

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.465

    GCN5 loses its activity following SAH. ( a ) The location of GCN5 in the neurons of sham or SAH rat were determined by co-staining with GCN5 and NeuN, scale bar=20 μ m. ( b ) The protein expression levels or mRNA levels of GCN5 were determined by WB or RT-PCR at 24 h after SAH. ( c ) At 24 h after SAH, the homogenized brain tissues were subjected to a histone extraction followed by WB to detect Ac-H3K9, Ac-H3K14, Ac-H3K27, H3, Ac-H4K12, H4, Ac-H2BK5 and H2B. ( d and e ) At 24 h after SAH, the homogenized brain tissues were subjected to IP and the partial precipitated complexes were subjected to WB and the left ones were subjected to in vitro HAT activity assay. The values are expressed as mean±S.E.M., * P
    Figure Legend Snippet: GCN5 loses its activity following SAH. ( a ) The location of GCN5 in the neurons of sham or SAH rat were determined by co-staining with GCN5 and NeuN, scale bar=20 μ m. ( b ) The protein expression levels or mRNA levels of GCN5 were determined by WB or RT-PCR at 24 h after SAH. ( c ) At 24 h after SAH, the homogenized brain tissues were subjected to a histone extraction followed by WB to detect Ac-H3K9, Ac-H3K14, Ac-H3K27, H3, Ac-H4K12, H4, Ac-H2BK5 and H2B. ( d and e ) At 24 h after SAH, the homogenized brain tissues were subjected to IP and the partial precipitated complexes were subjected to WB and the left ones were subjected to in vitro HAT activity assay. The values are expressed as mean±S.E.M., * P

    Techniques Used: Activity Assay, Staining, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, In Vitro, HAT Assay

    GCN5 loses its activity following potassium deprivation or glutamate exposure. ( a ) DIV 7 CGNs treated with 25K or 5K for 4 h, or glutamate (Glu, 100 μ M), were subjected to WB to detect Ac-H3K9, Ac-H3K14, Ac-H3K27, H3, Ac-H4K5, H4, Ac-H2BK5 and H2B. Con: control. ( b ) CGNs treated with 25K and 5K for two time durations 2 and 4 h were subjected to WB to detect Ac-H3K9, H3, E2F1, Egr-1 and Bim. ( c ) CGNs exposed to glutamate in 25K media for 8 h were subjected to WB to detect Ac-H3K9, H3, E2F1, Egr-1 and Bim. ( d and e ) CGNs treated with 25K and 5K for 4 h were subjected to IP with anti-GCN5 antibody and then to WB to detect the precipitated GCN5 (partial) and the left precipitated complexes were subjected to in vitro HAT activity. Mean±S.E.M., n =3, * P
    Figure Legend Snippet: GCN5 loses its activity following potassium deprivation or glutamate exposure. ( a ) DIV 7 CGNs treated with 25K or 5K for 4 h, or glutamate (Glu, 100 μ M), were subjected to WB to detect Ac-H3K9, Ac-H3K14, Ac-H3K27, H3, Ac-H4K5, H4, Ac-H2BK5 and H2B. Con: control. ( b ) CGNs treated with 25K and 5K for two time durations 2 and 4 h were subjected to WB to detect Ac-H3K9, H3, E2F1, Egr-1 and Bim. ( c ) CGNs exposed to glutamate in 25K media for 8 h were subjected to WB to detect Ac-H3K9, H3, E2F1, Egr-1 and Bim. ( d and e ) CGNs treated with 25K and 5K for 4 h were subjected to IP with anti-GCN5 antibody and then to WB to detect the precipitated GCN5 (partial) and the left precipitated complexes were subjected to in vitro HAT activity. Mean±S.E.M., n =3, * P

    Techniques Used: Activity Assay, Western Blot, In Vitro, HAT Assay

    13) Product Images from "A Mutation in Histone H2B Represents A New Class Of Oncogenic Driver"

    Article Title: A Mutation in Histone H2B Represents A New Class Of Oncogenic Driver

    Journal: Cancer discovery

    doi: 10.1158/2159-8290.CD-19-0393

    Survey of the most frequently found canonical histone mutations in cancer patient samples. A cross cancer mutation summary was performed using the cBioPortal to search a total of 41,738 non-redundant patient samples across all cancer types. The number of patients reported to have a missense mutation at each amino acid residue position across histone paralogs was graphed. A red line denotes the average number of mutations for each histone across all paralogs, and blue shaded regions indicate the first two standard deviations from the average. Location of the cumulative missense mutations found in: (A) 15 canonical H2A genes ( HIST1H2AA/B/C/D/E/G/H/I/J/K/L/M, HIST2H2AB/C, HIST3H2A ), (B) 18 canonical H2B genes ( HIST1H2BA/B/C/D/E/F/G/H/I/J/K/L/M/N/O, HIST2H2BE/F, HIST3H2BB ), (C) 12 canonical H3 genes ( HIST1H3A/B/C/D/E/F/G/H/I/J, HIST2H3D, HIST3H3 ), and (D) 14 canonical H4 genes ( HIST1H4A/B/C/D/E/F/G/H/I/J/K/L, HIST2H4A, HIST4H4 ) across all cancer patient samples. The Z scores indicate the most frequently found mutations (Z > 2) in each histone. Amino acids displayed in red were significantly more frequent in cBioPortal than the general SNP database.
    Figure Legend Snippet: Survey of the most frequently found canonical histone mutations in cancer patient samples. A cross cancer mutation summary was performed using the cBioPortal to search a total of 41,738 non-redundant patient samples across all cancer types. The number of patients reported to have a missense mutation at each amino acid residue position across histone paralogs was graphed. A red line denotes the average number of mutations for each histone across all paralogs, and blue shaded regions indicate the first two standard deviations from the average. Location of the cumulative missense mutations found in: (A) 15 canonical H2A genes ( HIST1H2AA/B/C/D/E/G/H/I/J/K/L/M, HIST2H2AB/C, HIST3H2A ), (B) 18 canonical H2B genes ( HIST1H2BA/B/C/D/E/F/G/H/I/J/K/L/M/N/O, HIST2H2BE/F, HIST3H2BB ), (C) 12 canonical H3 genes ( HIST1H3A/B/C/D/E/F/G/H/I/J, HIST2H3D, HIST3H3 ), and (D) 14 canonical H4 genes ( HIST1H4A/B/C/D/E/F/G/H/I/J/K/L, HIST2H4A, HIST4H4 ) across all cancer patient samples. The Z scores indicate the most frequently found mutations (Z > 2) in each histone. Amino acids displayed in red were significantly more frequent in cBioPortal than the general SNP database.

    Techniques Used: Mutagenesis

    Expression of mutant H2B in yeast destabilizes nucleosomes, deregulates gene expression and reduces nucleosome occupancy at the PHO5 promoter. WT or E79K H2B (analogous to human H2B-E76K) was expressed in S. Cerevisiae. (A) Yeast cells expressing H2B-E79K are temperature sensitive. Limiting dilutions of yeast expressing WT, E79A, E79Q or E79K were plated and incubated at 30°C or 37°C. Cell growth was evaluated after 1 day. (B) Yeast doubling time is significantly increased in cells expressing E79K-H2B at 37°C. (C) Time course of MNase sensitivity from spheroplasted yeast grown in rich media. M, marker. (D) Chromatin pellets were extracted with increasing concentrations of salt as indicated. Immuno-blotting of the soluble fraction was performed with antibody to H4. (E) Cells expressing WT, E79Q or E79K H2B were maintained in either rich media (YPDA) or phosphate-free media and expression of the phosphate-inducible PHO5 gene was measured by RT-PCR. (F) Nucleosome scanning assay of the PHO5 promoter from cells expressing either WT or E79K grown in rich media. Chromatin was digested with MNase, mononucleosomal DNA was purified and MNase protection was determined by qPCR. H2B occupancy at −2 nucleosome position of PHO5 is reduced in E79K cells as indicated by the arrow.
    Figure Legend Snippet: Expression of mutant H2B in yeast destabilizes nucleosomes, deregulates gene expression and reduces nucleosome occupancy at the PHO5 promoter. WT or E79K H2B (analogous to human H2B-E76K) was expressed in S. Cerevisiae. (A) Yeast cells expressing H2B-E79K are temperature sensitive. Limiting dilutions of yeast expressing WT, E79A, E79Q or E79K were plated and incubated at 30°C or 37°C. Cell growth was evaluated after 1 day. (B) Yeast doubling time is significantly increased in cells expressing E79K-H2B at 37°C. (C) Time course of MNase sensitivity from spheroplasted yeast grown in rich media. M, marker. (D) Chromatin pellets were extracted with increasing concentrations of salt as indicated. Immuno-blotting of the soluble fraction was performed with antibody to H4. (E) Cells expressing WT, E79Q or E79K H2B were maintained in either rich media (YPDA) or phosphate-free media and expression of the phosphate-inducible PHO5 gene was measured by RT-PCR. (F) Nucleosome scanning assay of the PHO5 promoter from cells expressing either WT or E79K grown in rich media. Chromatin was digested with MNase, mononucleosomal DNA was purified and MNase protection was determined by qPCR. H2B occupancy at −2 nucleosome position of PHO5 is reduced in E79K cells as indicated by the arrow.

    Techniques Used: Expressing, Mutagenesis, Incubation, Marker, Reverse Transcription Polymerase Chain Reaction, Purification, Real-time Polymerase Chain Reaction

    Expression of H2B-E76K alters growth properties and gene expression in human mammary epithelial cells. (A) Proliferation of MCF10A transduced with lentivirus expressing either WT or H2B-E76K was measured every 3 to 4 days. The results from three different infections are shown. Linear regression analysis was used to calculate statistical significance. (B) Heat maps depicting differential gene expression between MCF10A cells expressing either WT or H2B-E76K. (C and D) Bubble plots of gene ontology analysis identify biological processes significantly different in cells expressing H2B-E76K compared to cells expressing WT H2B. Bubble area is relative to the number of genes identified in each classification. Ontology of genes with increased (C) or decreased (D) expression in cells expressing H2B-E76K. (E) MCF10A cells expressing WT H2B or H2B-E76K and PIK3CA(H1047R) were grown in soft agar for 14 days and colonies counted.
    Figure Legend Snippet: Expression of H2B-E76K alters growth properties and gene expression in human mammary epithelial cells. (A) Proliferation of MCF10A transduced with lentivirus expressing either WT or H2B-E76K was measured every 3 to 4 days. The results from three different infections are shown. Linear regression analysis was used to calculate statistical significance. (B) Heat maps depicting differential gene expression between MCF10A cells expressing either WT or H2B-E76K. (C and D) Bubble plots of gene ontology analysis identify biological processes significantly different in cells expressing H2B-E76K compared to cells expressing WT H2B. Bubble area is relative to the number of genes identified in each classification. Ontology of genes with increased (C) or decreased (D) expression in cells expressing H2B-E76K. (E) MCF10A cells expressing WT H2B or H2B-E76K and PIK3CA(H1047R) were grown in soft agar for 14 days and colonies counted.

    Techniques Used: Expressing, Transduction

    Frequency of recurrent histone missense mutations in cancer. The cBioPortal was queried to determine the frequency of histone mutations in cancer patient samples at each primary site. (A) The frequency for any of the eighteen most significantly recurring histone missense mutations (Z score > 2) in each primary cancer site. (B) The frequency of missense mutations at either H2B glutamate 76 (H2B-E76), H4 aspartate (H4-D68) or H4 arginine (H4-R92), which destabilizes the histone octamer at a specific structural region, in cancers at each primary site. (C) The co-occurrence of the H2B-E76K mutation with mutations in the most common oncogenes found in each cancer type (for cancers with 3 or more patients with H2B-E76K) was assessed by a randomization test through 10,000 rounds of randomization.
    Figure Legend Snippet: Frequency of recurrent histone missense mutations in cancer. The cBioPortal was queried to determine the frequency of histone mutations in cancer patient samples at each primary site. (A) The frequency for any of the eighteen most significantly recurring histone missense mutations (Z score > 2) in each primary cancer site. (B) The frequency of missense mutations at either H2B glutamate 76 (H2B-E76), H4 aspartate (H4-D68) or H4 arginine (H4-R92), which destabilizes the histone octamer at a specific structural region, in cancers at each primary site. (C) The co-occurrence of the H2B-E76K mutation with mutations in the most common oncogenes found in each cancer type (for cancers with 3 or more patients with H2B-E76K) was assessed by a randomization test through 10,000 rounds of randomization.

    Techniques Used: Mutagenesis

    The E76K mutation in H2B destabilizes the histone octamer and fails to protect the nucleosome from nuclease treatment in vitro . (A) The H2B-E76K mutant was unable to form stable octamers in vitro . Recombinant human histones (H2A, H2B, H3, and H4) were mixed and histone octamers resolved from (H3/H4) 2 tetramers, H2A/H2B dimers and free histones by gel filtration chromatography. (B) Nucleosomes were reconstituted by mixing equimolar amounts of DNA (147bp) and octamers or in the case of E76K of tetramers and dimers (1:2 molar ratio) and resolved by Native PAGE. Nucleosomes containing H2B-E76K and E76Q have an altered migration pattern, intermediate between a tetrasome and a WT nucleosome. (C) Micrococcal nuclease (MNase) sensitivity assay performed on nucleosomes made with WT, E76Q and E76K H2B mutants shows more rapid digestion of E76K containing nucleosomes than those with WT H2B. A time course by gel (left) and densitometry quantification (right) of intact nucleosomes following MNase treatment. (D) The MNase susceptibility of E76K nucleosomes is distinct from nucleosomes formed only with tetrasomes.
    Figure Legend Snippet: The E76K mutation in H2B destabilizes the histone octamer and fails to protect the nucleosome from nuclease treatment in vitro . (A) The H2B-E76K mutant was unable to form stable octamers in vitro . Recombinant human histones (H2A, H2B, H3, and H4) were mixed and histone octamers resolved from (H3/H4) 2 tetramers, H2A/H2B dimers and free histones by gel filtration chromatography. (B) Nucleosomes were reconstituted by mixing equimolar amounts of DNA (147bp) and octamers or in the case of E76K of tetramers and dimers (1:2 molar ratio) and resolved by Native PAGE. Nucleosomes containing H2B-E76K and E76Q have an altered migration pattern, intermediate between a tetrasome and a WT nucleosome. (C) Micrococcal nuclease (MNase) sensitivity assay performed on nucleosomes made with WT, E76Q and E76K H2B mutants shows more rapid digestion of E76K containing nucleosomes than those with WT H2B. A time course by gel (left) and densitometry quantification (right) of intact nucleosomes following MNase treatment. (D) The MNase susceptibility of E76K nucleosomes is distinct from nucleosomes formed only with tetrasomes.

    Techniques Used: Mutagenesis, In Vitro, Recombinant, Filtration, Chromatography, Clear Native PAGE, Migration, Sensitive Assay

    H2B-E76K fundamentally alters chromatin structure and dynamics. (A) Micrococcal nuclease (MNase) assay with nuclei of MCF10A cells stably expressing either WT or H2B-E76K demonstrate that E76K expression significantly increases sensitivity to MNase. Digest efficiency was visualized by agarose gel. (B) Fluorescence recovery after photobleaching (FRAP) analysis demonstrates that H2B-E76K has significantly faster chromatin dynamics than WT. FRAP analysis was carried out after induction of either WT or E76K mutant H2B-GFP fusions for 5 or 35 days in MCF10A cells. Dashed lines represent cells expressing H2B-E76K, solid lines represent data from cells expressing inducible GFP-tagged WT H2B (n = 10 cells). (C) Representative FRAP assay pre-bleach, bleach and post-bleach images of nuclei expressing GFP-tagged WT or H2B-E76K indicate faster fluorescent recovery in cells expressing E76K. (D) FRAP analysis of histone H2A-GFP dynamics in MCF10A cells expressing only H2A-GFP (n = 20 cells) or both H2A-GFP and a mutant H2B E76K mCherry fusion (n = 25 cells) with standard deviation envelopes. Inset demonstrates co-expression of H2A GFP and E76K mCherry. (E) Representative pre-bleach, bleach and post-bleach images of H2A GFP in WT MCF10A cells or in cells co-expressing mCherry tagged H2B-E76K. (F) 100nm particles injected into the nucleus had significantly increased mean square displacement (MSD) over time in MCF10A cells stably expressing exogenous H2B-E76K (red) compared to cells expressing WT H2B (blue). N=68 (WT) and N=67 (E76K) cells were analyzed. Error bars represent SEM.
    Figure Legend Snippet: H2B-E76K fundamentally alters chromatin structure and dynamics. (A) Micrococcal nuclease (MNase) assay with nuclei of MCF10A cells stably expressing either WT or H2B-E76K demonstrate that E76K expression significantly increases sensitivity to MNase. Digest efficiency was visualized by agarose gel. (B) Fluorescence recovery after photobleaching (FRAP) analysis demonstrates that H2B-E76K has significantly faster chromatin dynamics than WT. FRAP analysis was carried out after induction of either WT or E76K mutant H2B-GFP fusions for 5 or 35 days in MCF10A cells. Dashed lines represent cells expressing H2B-E76K, solid lines represent data from cells expressing inducible GFP-tagged WT H2B (n = 10 cells). (C) Representative FRAP assay pre-bleach, bleach and post-bleach images of nuclei expressing GFP-tagged WT or H2B-E76K indicate faster fluorescent recovery in cells expressing E76K. (D) FRAP analysis of histone H2A-GFP dynamics in MCF10A cells expressing only H2A-GFP (n = 20 cells) or both H2A-GFP and a mutant H2B E76K mCherry fusion (n = 25 cells) with standard deviation envelopes. Inset demonstrates co-expression of H2A GFP and E76K mCherry. (E) Representative pre-bleach, bleach and post-bleach images of H2A GFP in WT MCF10A cells or in cells co-expressing mCherry tagged H2B-E76K. (F) 100nm particles injected into the nucleus had significantly increased mean square displacement (MSD) over time in MCF10A cells stably expressing exogenous H2B-E76K (red) compared to cells expressing WT H2B (blue). N=68 (WT) and N=67 (E76K) cells were analyzed. Error bars represent SEM.

    Techniques Used: Stable Transfection, Expressing, Agarose Gel Electrophoresis, Fluorescence, Mutagenesis, FRAP Assay, Standard Deviation, Injection

    14) Product Images from "Legacy Effect of Foxo1 in Pancreatic Endocrine Progenitors on Adult β-Cell Mass and Function"

    Article Title: Legacy Effect of Foxo1 in Pancreatic Endocrine Progenitors on Adult β-Cell Mass and Function

    Journal: Diabetes

    doi: 10.2337/db14-1696

    A self-replicating pool of endocrine progenitors in adult PKO mice. A : Quantification of Ki67 + /insulin + cells by flow cytometry in mice of the indicated ages and genotypes. We normalized the number of double-positive cells by the total number of insulin + cells per pancreas ( n = 6 each). B : Quantification of Ki67 + /insulin + cells by double immunohistochemistry in 12-month-old mice of the indicated genotype by manual counting, normalized by total number of insulin + cells per pancreas ( n = 4 each). C : Double immunohistochemistry with anti-Ki67 (dark brown) and anti-insulin antibodies (light brown) on pancreatic sections of 7-day-old mice ( n = 4). Insets show representative insulin + and/or Ki67 + cells. D : Immunohistochemistry with anti-Ki67 antibody (dark brown) on pancreas sections of 15-month-old mice ( n = 4 each). Insets represent Ki67 + cells in islets. E : qPCR analysis of a panel of cell cycle regulatory genes in purified islets from control and PKO mice ( n = 6 each). F : Immunofluorescence with anti-Neurog3 (green) and anti-H2B (purple) in pancreas sections from 3-month-old control and PKO mice ( n = 4 each). Inset shows Neurog3 + /H2B + cells. G : Immunofluorescence with anti-Neurog3 (green) and anti-Ki67 (red) in pancreas sections from 15-month-old mice ( n = 4 each). Insets show Neurog3 + and Neurog3 + /Ki67 + cells. F and G : A dashed line is used to outline the islet. Data show means ± SEM. * P
    Figure Legend Snippet: A self-replicating pool of endocrine progenitors in adult PKO mice. A : Quantification of Ki67 + /insulin + cells by flow cytometry in mice of the indicated ages and genotypes. We normalized the number of double-positive cells by the total number of insulin + cells per pancreas ( n = 6 each). B : Quantification of Ki67 + /insulin + cells by double immunohistochemistry in 12-month-old mice of the indicated genotype by manual counting, normalized by total number of insulin + cells per pancreas ( n = 4 each). C : Double immunohistochemistry with anti-Ki67 (dark brown) and anti-insulin antibodies (light brown) on pancreatic sections of 7-day-old mice ( n = 4). Insets show representative insulin + and/or Ki67 + cells. D : Immunohistochemistry with anti-Ki67 antibody (dark brown) on pancreas sections of 15-month-old mice ( n = 4 each). Insets represent Ki67 + cells in islets. E : qPCR analysis of a panel of cell cycle regulatory genes in purified islets from control and PKO mice ( n = 6 each). F : Immunofluorescence with anti-Neurog3 (green) and anti-H2B (purple) in pancreas sections from 3-month-old control and PKO mice ( n = 4 each). Inset shows Neurog3 + /H2B + cells. G : Immunofluorescence with anti-Neurog3 (green) and anti-Ki67 (red) in pancreas sections from 15-month-old mice ( n = 4 each). Insets show Neurog3 + and Neurog3 + /Ki67 + cells. F and G : A dashed line is used to outline the islet. Data show means ± SEM. * P

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Immunohistochemistry, Real-time Polymerase Chain Reaction, Purification, Immunofluorescence

    15) Product Images from "Astrocyte Reactivity Following Blast Exposure Involves Aberrant Histone Acetylation"

    Article Title: Astrocyte Reactivity Following Blast Exposure Involves Aberrant Histone Acetylation

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2016.00064

    (A) Western blot images created by Compass Software (ProteinSimple, Santa Clara, CA, USA) representing modified and total histone expression in the prefrontal cortex (PFC) for each treatment group. No differences in total histone expression was observed for any histone protein. (B) Levels of acetyl-H2b (AH2b), acetyl-H3 (AH3), and acetyl-H4 (AH4) were significantly decreased compared to sham. No differences were observed between injury groups. *Indicates p
    Figure Legend Snippet: (A) Western blot images created by Compass Software (ProteinSimple, Santa Clara, CA, USA) representing modified and total histone expression in the prefrontal cortex (PFC) for each treatment group. No differences in total histone expression was observed for any histone protein. (B) Levels of acetyl-H2b (AH2b), acetyl-H3 (AH3), and acetyl-H4 (AH4) were significantly decreased compared to sham. No differences were observed between injury groups. *Indicates p

    Techniques Used: Western Blot, Software, Modification, Expressing

    16) Product Images from "Condensed mitotic chromatin is accessible to transcription factors and chromatin structural proteins"

    Article Title: Condensed mitotic chromatin is accessible to transcription factors and chromatin structural proteins

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200407182

    FRAP analyses of core histones at all phases of mitosis. Quantification of the fluorescence recoveries through changes in RFI over time for GFP-H2A, H2B-GFP, H3-GFP, and H4-GFP, correspondingly. Error bars represent averages from 10 cells ± SD.
    Figure Legend Snippet: FRAP analyses of core histones at all phases of mitosis. Quantification of the fluorescence recoveries through changes in RFI over time for GFP-H2A, H2B-GFP, H3-GFP, and H4-GFP, correspondingly. Error bars represent averages from 10 cells ± SD.

    Techniques Used: Fluorescence

    FRAP analyses of H2B-GFP at all phases of mitosis. Cells were imaged before and during recovery after bleaching of chromosome area. For each M phase cell, top panel represents original images, bottom panel represents pseudocolored images. Bar, 10 μM.
    Figure Legend Snippet: FRAP analyses of H2B-GFP at all phases of mitosis. Cells were imaged before and during recovery after bleaching of chromosome area. For each M phase cell, top panel represents original images, bottom panel represents pseudocolored images. Bar, 10 μM.

    Techniques Used:

    17) Product Images from "EPC1/TIP60-Mediated Histone Acetylation Facilitates Spermiogenesis in Mice"

    Article Title: EPC1/TIP60-Mediated Histone Acetylation Facilitates Spermiogenesis in Mice

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00082-17

    Impaired histone hyperacetylation and TNP2 incorporation of Epc1 -KO spermatids. (A) Defects in histone hyperacetylation in Epc1 -KO spermatids. Paraffin sections were indirectly immunostained with anti-acetylated histone H4 (a and b), H2A (c and d), H2B (e and f), or H3 (g and h) antibody in Epc1 -KO (−/−) spermatids and littermate heterozygotes (+/−). SCP3 antibody was used for detection of leptotene (arrows) and pachytene (arrowheads) spermatocytes and staging of seminiferous tubules (a, a′, b, and b′). In the presumed stage 9 seminiferous tubules of Epc1 -KO mice; leptotene and pachytene spermatocytes were normally layered from basal membrane to seminiferous tubule lumen, but ESs containing hyperacetylated histone were hardly detectable. In addition, a layer of round spermatids was expanded. Scale bars, 50 μm. (B) Reduced histone acetylation levels in Epc1 -KO germ cells. Acetylation levels of histone H4 (AcH4) and H2A (AcH2A) in adult germ cells were compared between Epc1 -KO (−/−) and wild-type littermates (+/+) by IB analysis. (C) Excess accumulation of RSs demarcated by ubiquitinated histone H2A. Expression of ubiquitinated histone H2A (uH2A) in Epc1 -KO seminiferous tubules was examined by IF analysis. Arrowheads indicate RSs. Scale bar, 10 μm. (D) Increased histone ubiquitination levels in Epc1 -KO germ cells. Ubiquitination levels of histone H2A (uH2A) and H2B (uH2B) were examined by IB analysis in adult germ cells. (E) Partial restoration of elongating spermatids in Epc1 -KO seminiferous tubules. Paraffin sections were immunostained for acetylated histone H2B and transition protein 2 (TNP2) in Epc1 -KO mice (−/−) and littermate heterozygotes (+/−). A cluster of Epc1 -KO spermatids exhibited a partial restoration of histone H2B acetylation (AcH2B) and a simultaneous incorporation of TNP2 along with an elongating morphology. Enlarged views for the boxed regions in panels a to h are shown in panels a′ to h′, respectively. Scale bars, 50 μm (a) and 5 μm (a′). (F) Partial restoration of histone acetylation levels in Epc1 -KO spermatids with an elongating morphology. A spread preparation of spermatids (left) was used to examine morphology, PNA distribution, and histone H4 acetylation levels (AcH4). Representative elongating Epc1 -KO spermatids that escaped from maturation arrest are shown. Scale bar, 10 μm. Relative quantification of histone H4 acetylation levels in elongating Epc1 -KO ( n = 32) and wild-type ( n = 20) ESs was performed by using ImageJ, and the results are summarized by box plots (right). The boxes represent the interquartile deviation. (G) The impact of trichostatin A (TSA) treatment on histone H4 acetylation and TNP2 incorporation in Epc1 -KO spermatids. Epc1 -KO germ cells were cultured for 3 days with (+) or without (−) TSA and subjected to IB analyses for acetylated histone H4, TNP2, and β-actin. TNP2/β-actin ratios based on respective band intensities are shown at the bottom, in which the ratio in untreated Epc1 -KO germ cells was set as 1.
    Figure Legend Snippet: Impaired histone hyperacetylation and TNP2 incorporation of Epc1 -KO spermatids. (A) Defects in histone hyperacetylation in Epc1 -KO spermatids. Paraffin sections were indirectly immunostained with anti-acetylated histone H4 (a and b), H2A (c and d), H2B (e and f), or H3 (g and h) antibody in Epc1 -KO (−/−) spermatids and littermate heterozygotes (+/−). SCP3 antibody was used for detection of leptotene (arrows) and pachytene (arrowheads) spermatocytes and staging of seminiferous tubules (a, a′, b, and b′). In the presumed stage 9 seminiferous tubules of Epc1 -KO mice; leptotene and pachytene spermatocytes were normally layered from basal membrane to seminiferous tubule lumen, but ESs containing hyperacetylated histone were hardly detectable. In addition, a layer of round spermatids was expanded. Scale bars, 50 μm. (B) Reduced histone acetylation levels in Epc1 -KO germ cells. Acetylation levels of histone H4 (AcH4) and H2A (AcH2A) in adult germ cells were compared between Epc1 -KO (−/−) and wild-type littermates (+/+) by IB analysis. (C) Excess accumulation of RSs demarcated by ubiquitinated histone H2A. Expression of ubiquitinated histone H2A (uH2A) in Epc1 -KO seminiferous tubules was examined by IF analysis. Arrowheads indicate RSs. Scale bar, 10 μm. (D) Increased histone ubiquitination levels in Epc1 -KO germ cells. Ubiquitination levels of histone H2A (uH2A) and H2B (uH2B) were examined by IB analysis in adult germ cells. (E) Partial restoration of elongating spermatids in Epc1 -KO seminiferous tubules. Paraffin sections were immunostained for acetylated histone H2B and transition protein 2 (TNP2) in Epc1 -KO mice (−/−) and littermate heterozygotes (+/−). A cluster of Epc1 -KO spermatids exhibited a partial restoration of histone H2B acetylation (AcH2B) and a simultaneous incorporation of TNP2 along with an elongating morphology. Enlarged views for the boxed regions in panels a to h are shown in panels a′ to h′, respectively. Scale bars, 50 μm (a) and 5 μm (a′). (F) Partial restoration of histone acetylation levels in Epc1 -KO spermatids with an elongating morphology. A spread preparation of spermatids (left) was used to examine morphology, PNA distribution, and histone H4 acetylation levels (AcH4). Representative elongating Epc1 -KO spermatids that escaped from maturation arrest are shown. Scale bar, 10 μm. Relative quantification of histone H4 acetylation levels in elongating Epc1 -KO ( n = 32) and wild-type ( n = 20) ESs was performed by using ImageJ, and the results are summarized by box plots (right). The boxes represent the interquartile deviation. (G) The impact of trichostatin A (TSA) treatment on histone H4 acetylation and TNP2 incorporation in Epc1 -KO spermatids. Epc1 -KO germ cells were cultured for 3 days with (+) or without (−) TSA and subjected to IB analyses for acetylated histone H4, TNP2, and β-actin. TNP2/β-actin ratios based on respective band intensities are shown at the bottom, in which the ratio in untreated Epc1 -KO germ cells was set as 1.

    Techniques Used: Mouse Assay, Expressing, Cell Culture

    18) Product Images from "Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response"

    Article Title: Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1814965116

    Chromatin architecture demarcates the repair locus. ( A ) FLIM-FRET maps acquired in HeLa H2B-2FP cells 10 min before and 0, 30, and 60 min after microirradiation ( Upper ) and expanded images of the DSB ROI ( Lower ). ( B ) eGFP-53BP1 intensity images acquired in HeLa cells 10 min before and 0, 30, and 60 min after microirradiation ( Upper ) and expanded images of the DSB ROI ( Lower ). ( C ) Correlation of compact chromatin foci localization along the horizontal axis as a function of time ( Top ; image from 0 min is shown), with 53BP1 localization ( Middle ; image from 0 min is shown), and mobility in terms of time delay ( Bottom ). eGFP-53BP1 localization and mobility are averaged along the horizontal axes (green plots). Red dashed box indicates laser microirradiation ROI. ( D ) Comparison of compact chromatin foci localization ( Top ), eGFP-53BP1 localization ( Middle ), and eGFP-53BP1 mobility ( Bottom ) in untreated HeLa cells (blue curve), KU-55933–treated HeLa cells (green curve), or RNF8 KO cells (red curve) 10 min before and 0, 30, and 60 min after microirradiation. Representative example of n = 3 shown. (Scale bars, 5 μm.)
    Figure Legend Snippet: Chromatin architecture demarcates the repair locus. ( A ) FLIM-FRET maps acquired in HeLa H2B-2FP cells 10 min before and 0, 30, and 60 min after microirradiation ( Upper ) and expanded images of the DSB ROI ( Lower ). ( B ) eGFP-53BP1 intensity images acquired in HeLa cells 10 min before and 0, 30, and 60 min after microirradiation ( Upper ) and expanded images of the DSB ROI ( Lower ). ( C ) Correlation of compact chromatin foci localization along the horizontal axis as a function of time ( Top ; image from 0 min is shown), with 53BP1 localization ( Middle ; image from 0 min is shown), and mobility in terms of time delay ( Bottom ). eGFP-53BP1 localization and mobility are averaged along the horizontal axes (green plots). Red dashed box indicates laser microirradiation ROI. ( D ) Comparison of compact chromatin foci localization ( Top ), eGFP-53BP1 localization ( Middle ), and eGFP-53BP1 mobility ( Bottom ) in untreated HeLa cells (blue curve), KU-55933–treated HeLa cells (green curve), or RNF8 KO cells (red curve) 10 min before and 0, 30, and 60 min after microirradiation. Representative example of n = 3 shown. (Scale bars, 5 μm.)

    Techniques Used:

    ATM and RNF8 regulate chromatin architecture in the DDR. ( A ) FLIM-FRET maps of HeLa H2B-2FP , HeLa H2B-2FP treated with KU-55933, or HeLa H2B-2FP RNF8 KO cells 10 min before and 1, 3, and 6 h after microirradiation. ( B and C ) Fraction of compacted chromatin pixels within ( B ) and outside ( C ) of the NIR irradiation site in HeLa H2B-2FP (blue curve; n = 10), KU-55933-treated (green curve; n = 6), or RNF8 KO cells (red curve; n = 3; mean ± SEM). ( D ) Longevity maps of compact chromatin foci from the cells in A . ( E ) Quantification of compact chromatin foci stability ( Left ) throughout the DDR ( Right ) in NIR laser-treated HeLa H2B-2FP , KU-55933–treated HeLa H2B-2FP , and HeLa H2B-2FP RNF8 KO cells ( n as indicated above, mean ± SEM). ( F ) Chromatin compaction size map derived by PLICS from the cells shown in A . ( G and H ) Change in the mean size of ( G ) and distance between ( H ) compacted chromatin foci during the DDR in NIR laser-treated HeLa H2B-2FP , KU-55933–treated HeLa H2B-2FP , and HeLa H2B-2FP RNF8 KO cells ( n as indicated above, mean ± SEM). (Scale bars, 5 μm.)
    Figure Legend Snippet: ATM and RNF8 regulate chromatin architecture in the DDR. ( A ) FLIM-FRET maps of HeLa H2B-2FP , HeLa H2B-2FP treated with KU-55933, or HeLa H2B-2FP RNF8 KO cells 10 min before and 1, 3, and 6 h after microirradiation. ( B and C ) Fraction of compacted chromatin pixels within ( B ) and outside ( C ) of the NIR irradiation site in HeLa H2B-2FP (blue curve; n = 10), KU-55933-treated (green curve; n = 6), or RNF8 KO cells (red curve; n = 3; mean ± SEM). ( D ) Longevity maps of compact chromatin foci from the cells in A . ( E ) Quantification of compact chromatin foci stability ( Left ) throughout the DDR ( Right ) in NIR laser-treated HeLa H2B-2FP , KU-55933–treated HeLa H2B-2FP , and HeLa H2B-2FP RNF8 KO cells ( n as indicated above, mean ± SEM). ( F ) Chromatin compaction size map derived by PLICS from the cells shown in A . ( G and H ) Change in the mean size of ( G ) and distance between ( H ) compacted chromatin foci during the DDR in NIR laser-treated HeLa H2B-2FP , KU-55933–treated HeLa H2B-2FP , and HeLa H2B-2FP RNF8 KO cells ( n as indicated above, mean ± SEM). (Scale bars, 5 μm.)

    Techniques Used: Irradiation, Derivative Assay

    ICS applied to a phasor FLIM-FRET map measures nuclear-wide compact chromatin stability, size, and spacing. ( A – C ) Foci map. ( A ) FLIM-FRET map from an unirradiated HeLa H2B-2FP nucleus. ( B and C ) Pixel coordinates of the high FRET state (16–21% FRET) can be extracted from the phasor plot ( B ) to produce a localization map of compact chromatin ( C ). ( D – F ) Longevity analysis. ( D ) Localization map of compacted chromatin from the HeLa H2B-2FP cell in A at 0, 10, and 20 min (as detected by FRET). ( E ) Schematic of longevity analysis: Averaging three binary images gives rise to a heat map of pixel longevity, which contains structural information that is not evident in the source images and can be used to quantify the stability of detected structures. ( F ) Longevity map of the compacted chromatin foci detected and tracked in D , with digital enlargement shown for a ROI that contains foci present for 10 min (green pixels) and 20 min (red pixels). The fraction of foci persistence across the time course is calculated as a measure of overall chromatin network stability. ( G – I ) PLICS analysis. ( G ) A localization map of compacted chromatin from an unirradiated HeLa H2B-2FP nucleus as detected by FLIM-FRET. ( H ) Schematic of PLICS analysis: In a binary image showing different-sized structures, we can calculate localized 2D spatial correlation functions using an m × m matrix and by collapsing them into 1D correlation profiles. The resulting decay is characteristic of the size of a structure within each m × m matrix. By transforming each decay into phasor coordinates ( g , s ), we can graphically pseudocolor each pixel according to size. ( I ) Size map of compacted chromatin foci detected in G and PLICS/iPLICS analysis of the average size (305 nm) and spacing (485 nm). (Scale bars, 5 μm.)
    Figure Legend Snippet: ICS applied to a phasor FLIM-FRET map measures nuclear-wide compact chromatin stability, size, and spacing. ( A – C ) Foci map. ( A ) FLIM-FRET map from an unirradiated HeLa H2B-2FP nucleus. ( B and C ) Pixel coordinates of the high FRET state (16–21% FRET) can be extracted from the phasor plot ( B ) to produce a localization map of compact chromatin ( C ). ( D – F ) Longevity analysis. ( D ) Localization map of compacted chromatin from the HeLa H2B-2FP cell in A at 0, 10, and 20 min (as detected by FRET). ( E ) Schematic of longevity analysis: Averaging three binary images gives rise to a heat map of pixel longevity, which contains structural information that is not evident in the source images and can be used to quantify the stability of detected structures. ( F ) Longevity map of the compacted chromatin foci detected and tracked in D , with digital enlargement shown for a ROI that contains foci present for 10 min (green pixels) and 20 min (red pixels). The fraction of foci persistence across the time course is calculated as a measure of overall chromatin network stability. ( G – I ) PLICS analysis. ( G ) A localization map of compacted chromatin from an unirradiated HeLa H2B-2FP nucleus as detected by FLIM-FRET. ( H ) Schematic of PLICS analysis: In a binary image showing different-sized structures, we can calculate localized 2D spatial correlation functions using an m × m matrix and by collapsing them into 1D correlation profiles. The resulting decay is characteristic of the size of a structure within each m × m matrix. By transforming each decay into phasor coordinates ( g , s ), we can graphically pseudocolor each pixel according to size. ( I ) Size map of compacted chromatin foci detected in G and PLICS/iPLICS analysis of the average size (305 nm) and spacing (485 nm). (Scale bars, 5 μm.)

    Techniques Used:

    Phasor approach to FLIM-FRET analysis of chromatin compaction. ( A ) HeLa H2B-2FP nucleus coexpressing H2B-eGFP and H2B-mCherry (H2B-mCh). ( B ) Graphical depiction of how increasing nucleosome proximity leads to increased FRET between fluorescent histones. ( C ) Graphical depiction of phasor transformation of HeLa H2B-2FP FLIM-FRET data. ( C , Left ) Fluorescence lifetime of H2B-eGFP reports on the degree of FRET interaction in each pixel. Each line represents the fluorescent lifetime from a different pixel. ( C , Right ) These data when phasor-transformed give rise to phasor coordinates ( s , g ). The donor phasor is right-shifted to shorter fluorescent lifetimes depending on the efficiency of FRET interaction. In HeLa H2B-2FP , decreasing lifetime and increasing FRET corresponds to more compact chromatin. ( D ) Untreated, TSA-treated, or Actinomycin D (Act D)-treated HeLa H2B-2FP nuclei, shown in the H2B-eGFP channel. ( E ) Combined phasor distribution of H2B-eGFP fluorescence lifetime from all conditions shown in D with the theoretical FRET trajectory superimposed to determine the range of FRET efficiencies in HeLa H2B-2FP . The linear combination of unquenched donor and background cellular autofluorescence (teal–bright green) (defined in SI Appendix , Fig. S3 ) follows a distinct trajectory from FRET (teal–red). ( F ) Fraction of pixels in a compact (red) vs. open (teal) chromatin state in control (Cntrl), TSA-, and Act D-treated cells. ( G ) Pseudocolored chromatin compaction maps of the cells in D according to the palette defined in the phasor plot data in E . (Scale bars, 5 μm.)
    Figure Legend Snippet: Phasor approach to FLIM-FRET analysis of chromatin compaction. ( A ) HeLa H2B-2FP nucleus coexpressing H2B-eGFP and H2B-mCherry (H2B-mCh). ( B ) Graphical depiction of how increasing nucleosome proximity leads to increased FRET between fluorescent histones. ( C ) Graphical depiction of phasor transformation of HeLa H2B-2FP FLIM-FRET data. ( C , Left ) Fluorescence lifetime of H2B-eGFP reports on the degree of FRET interaction in each pixel. Each line represents the fluorescent lifetime from a different pixel. ( C , Right ) These data when phasor-transformed give rise to phasor coordinates ( s , g ). The donor phasor is right-shifted to shorter fluorescent lifetimes depending on the efficiency of FRET interaction. In HeLa H2B-2FP , decreasing lifetime and increasing FRET corresponds to more compact chromatin. ( D ) Untreated, TSA-treated, or Actinomycin D (Act D)-treated HeLa H2B-2FP nuclei, shown in the H2B-eGFP channel. ( E ) Combined phasor distribution of H2B-eGFP fluorescence lifetime from all conditions shown in D with the theoretical FRET trajectory superimposed to determine the range of FRET efficiencies in HeLa H2B-2FP . The linear combination of unquenched donor and background cellular autofluorescence (teal–bright green) (defined in SI Appendix , Fig. S3 ) follows a distinct trajectory from FRET (teal–red). ( F ) Fraction of pixels in a compact (red) vs. open (teal) chromatin state in control (Cntrl), TSA-, and Act D-treated cells. ( G ) Pseudocolored chromatin compaction maps of the cells in D according to the palette defined in the phasor plot data in E . (Scale bars, 5 μm.)

    Techniques Used: Transformation Assay, Fluorescence, Activated Clotting Time Assay

    A compact chromatin border forms around the lesion site within the first 30 min of the DDR. ( A ) FLIM-FRET maps acquired in a HeLa H2B-2FP cell during the first 30 min after NIR irradiation. ( B ) Longevity maps of compact chromatin foci taken at 10-min intervals over a 20-min duration. ( C ) Quantification of the number of stable compact chromatin pixels across multiple cells ( n = 10, mean ± SEM) in the first hour following NIR-induced DSBs (dashed line represents average number of stable compact chromatin pixels in an unperturbed cell). ( D ) Chromatin compaction size maps derived by PLICS analysis 0–30 min after DSB induction. ( E ) PLICS analysis of the histogram of sizes induced by DSBs in the data presented in D . ( F ) iPLICS analysis of the histogram of distance changes induced by DSBs in the data presented in D . ( G ) Change in mean size and distance between compacted chromatin foci as a function of time during the DDR ( n = 10 cells, mean ± SEM). (Scale bars, 5 μm.)
    Figure Legend Snippet: A compact chromatin border forms around the lesion site within the first 30 min of the DDR. ( A ) FLIM-FRET maps acquired in a HeLa H2B-2FP cell during the first 30 min after NIR irradiation. ( B ) Longevity maps of compact chromatin foci taken at 10-min intervals over a 20-min duration. ( C ) Quantification of the number of stable compact chromatin pixels across multiple cells ( n = 10, mean ± SEM) in the first hour following NIR-induced DSBs (dashed line represents average number of stable compact chromatin pixels in an unperturbed cell). ( D ) Chromatin compaction size maps derived by PLICS analysis 0–30 min after DSB induction. ( E ) PLICS analysis of the histogram of sizes induced by DSBs in the data presented in D . ( F ) iPLICS analysis of the histogram of distance changes induced by DSBs in the data presented in D . ( G ) Change in mean size and distance between compacted chromatin foci as a function of time during the DDR ( n = 10 cells, mean ± SEM). (Scale bars, 5 μm.)

    Techniques Used: Irradiation, Derivative Assay

    FLIM-FRET analysis of chromatin compaction reveals chromatin architectural changes during the DDR. ( A and B ) Time series of H2B-eGFP fluorescence intensity images ( A ) and lifetime maps ( B ) acquired in a HeLa H2B-2FP 10 min before and at hourly intervals after NIR irradiation. The white square indicates the NIR laser-treated locus. ( C ) Digital enlargement of the DNA damage site selected in B and the corresponding time series of lifetime maps within this ROI. ( D ) Masks selected for analysis of the number of pixels in a compacted (high-FRET) vs. noncompacted (low-FRET) state at the DNA damage site vs. outside this ROI. ( E and F ) Fraction of pixels within ( E ) and outside ( F ) of the NIR-irradiated ROI that are in a compacted state during the DDR (red curve) vs. an unperturbed cell (black curve) ( n = 10 cells, mean ± SEM). (Scale bars, 5 μm.)
    Figure Legend Snippet: FLIM-FRET analysis of chromatin compaction reveals chromatin architectural changes during the DDR. ( A and B ) Time series of H2B-eGFP fluorescence intensity images ( A ) and lifetime maps ( B ) acquired in a HeLa H2B-2FP 10 min before and at hourly intervals after NIR irradiation. The white square indicates the NIR laser-treated locus. ( C ) Digital enlargement of the DNA damage site selected in B and the corresponding time series of lifetime maps within this ROI. ( D ) Masks selected for analysis of the number of pixels in a compacted (high-FRET) vs. noncompacted (low-FRET) state at the DNA damage site vs. outside this ROI. ( E and F ) Fraction of pixels within ( E ) and outside ( F ) of the NIR-irradiated ROI that are in a compacted state during the DDR (red curve) vs. an unperturbed cell (black curve) ( n = 10 cells, mean ± SEM). (Scale bars, 5 μm.)

    Techniques Used: Fluorescence, Irradiation

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    Cell Signaling Technology Inc control h2b rfp vector
    Quantification of endogenous SMAD1/5 activity and Id gene expression in function of the mode of division. (A and B) Representative sections of HH18 neural tubes stained for the active form of SMAD1/5/8 (pS158) and pH3 (A) or lamin B1 (B). (C–H) Analysis of the endogenous SMAD1/5/8 activity in the distinct modes of divisions. Neural tube sections were stained for pS158 24 hpe of HH14 embryos with a combination of the pSox2:EGFP and the control <t>H2B-RFP</t> vector (C), the pTis21:RFP and the control H2B-GFP vector (E), or both pSox2:EGFP and pTis21:RFP reporters (G). The intensity of the mean nuclear pS158 staining was measured in pSox2 − and pSox2 + mitoses (D), in pTis21 − and pTis21 + mitoses (F), or in mitotic GFP + ;RFP − (PP), GFP + ;RFP + (PN), and GFP − ;RFP + (NN) progenitors (H). (I) Illustration of the methodology used to analyze the levels of Id1/2/3 transcripts expressed by the PP, PN, and NN subpopulations. (J) Semiquantitative PCR analysis of the mRNA levels of Id1, Id2, and Id3 transcripts expressed by GFP + ;RFP − (PP), GFP + ;RFP + (PN), and GFP − ;RFP + (NN) cells. a.u., arbitrary unit; EP, electroporation. Error bars show means ± SEM. *, P
    Control H2b Rfp Vector, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Integrin α6β4 signaling promotes recruitment of p53 and 53BP1 to chromatin in response to cisplatin treatment. BT549 cells (EV and β4) plated on laminin-1 and treated with 10 μM cisplatin for 24 hrs were harvested for cytosolic or nuclear fractions and immuno-blotted for phospho-p53 using actin and p84 as controls (A) or for immunocytochemistry staining for phospho-p53 S15 and phospho-53BP1 S1778 as indicated (B, scale bars, 20µm). (C) Subcellular protein fractionation was performed on cells treated as in (A) and noted fractions were immunoblotted with DDR proteins as indicated. Tubulin was used as the marker for total and cytosolic protein, Histone <t>H2B</t> and p84 were used as the markers for nuclear fractions.
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    Demonstration of antibody specificity to <t>D-isoAsp-H2B</t> (A) Western blots of histones from pcmt1 WT or KO mouse brain using a commercial pan-H2B antibody (H2B, top row), and our custom antibody made against the D-isoAsp-H2B synthetic peptide (H2B*, bottom row). Because the same histone preps were used for both blots, the top row serves as a loading control for the bottom row. (B) There is little difference (p = 0.235) between the WT and KO brain extracts when the pan-H2B antibody is used for blotting, but a large and significant (p = 0.003) difference when the H2B* antibody is used. Panel C shows that, when normalized to the pan-H2B signals, immuno-reactivity with the H2B* antibody is 2X stronger (p = 0.001) in the WT vs. KO extracts.
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    Cell Signaling Technology Inc acetyl h2b
    (A) Western blot images created by Compass Software (ProteinSimple, Santa Clara, CA, USA) representing modified and total histone expression in the prefrontal cortex (PFC) for each treatment group. No differences in total histone expression was observed for any histone protein. (B) Levels of <t>acetyl-H2b</t> (AH2b), acetyl-H3 (AH3), and acetyl-H4 (AH4) were significantly decreased compared to sham. No differences were observed between injury groups. *Indicates p
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    Quantification of endogenous SMAD1/5 activity and Id gene expression in function of the mode of division. (A and B) Representative sections of HH18 neural tubes stained for the active form of SMAD1/5/8 (pS158) and pH3 (A) or lamin B1 (B). (C–H) Analysis of the endogenous SMAD1/5/8 activity in the distinct modes of divisions. Neural tube sections were stained for pS158 24 hpe of HH14 embryos with a combination of the pSox2:EGFP and the control H2B-RFP vector (C), the pTis21:RFP and the control H2B-GFP vector (E), or both pSox2:EGFP and pTis21:RFP reporters (G). The intensity of the mean nuclear pS158 staining was measured in pSox2 − and pSox2 + mitoses (D), in pTis21 − and pTis21 + mitoses (F), or in mitotic GFP + ;RFP − (PP), GFP + ;RFP + (PN), and GFP − ;RFP + (NN) progenitors (H). (I) Illustration of the methodology used to analyze the levels of Id1/2/3 transcripts expressed by the PP, PN, and NN subpopulations. (J) Semiquantitative PCR analysis of the mRNA levels of Id1, Id2, and Id3 transcripts expressed by GFP + ;RFP − (PP), GFP + ;RFP + (PN), and GFP − ;RFP + (NN) cells. a.u., arbitrary unit; EP, electroporation. Error bars show means ± SEM. *, P

    Journal: The Journal of Cell Biology

    Article Title: The strength of SMAD1/5 activity determines the mode of stem cell division in the developing spinal cord

    doi: 10.1083/jcb.201307031

    Figure Lengend Snippet: Quantification of endogenous SMAD1/5 activity and Id gene expression in function of the mode of division. (A and B) Representative sections of HH18 neural tubes stained for the active form of SMAD1/5/8 (pS158) and pH3 (A) or lamin B1 (B). (C–H) Analysis of the endogenous SMAD1/5/8 activity in the distinct modes of divisions. Neural tube sections were stained for pS158 24 hpe of HH14 embryos with a combination of the pSox2:EGFP and the control H2B-RFP vector (C), the pTis21:RFP and the control H2B-GFP vector (E), or both pSox2:EGFP and pTis21:RFP reporters (G). The intensity of the mean nuclear pS158 staining was measured in pSox2 − and pSox2 + mitoses (D), in pTis21 − and pTis21 + mitoses (F), or in mitotic GFP + ;RFP − (PP), GFP + ;RFP + (PN), and GFP − ;RFP + (NN) progenitors (H). (I) Illustration of the methodology used to analyze the levels of Id1/2/3 transcripts expressed by the PP, PN, and NN subpopulations. (J) Semiquantitative PCR analysis of the mRNA levels of Id1, Id2, and Id3 transcripts expressed by GFP + ;RFP − (PP), GFP + ;RFP + (PN), and GFP − ;RFP + (NN) cells. a.u., arbitrary unit; EP, electroporation. Error bars show means ± SEM. *, P

    Article Snippet: HH14 embryos electroporated with either pSox2:EGFP and a control H2B-RFP vector, pTis21:RFP and a control H2B-GFP vector, or pSox2:EGFP and pTis21:RFP were recovered at 24 hpe and processed for immunohistochemistry using the phospho-SMAD1/5/8 antibody (pS158; Cell Signaling Technology).

    Techniques: Activity Assay, Expressing, Staining, Plasmid Preparation, Polymerase Chain Reaction, Electroporation

    SMAD1/5 inhibition triggers premature differentiation in a cell-autonomous manner. (A) Proportions of electroporated cells differentiated into neurons (H2B-RFP + ;HuC/D + ) obtained after electroporation of HH14 embryos with control (Ctrl) and Smad1/5 shRNA (sh-S1 and sh-S5) constructs, in sections of neural tubes recovered at the times indicated (hpe). (B) Representative sections stained for HuC/D expression 24 h after coelectroporation with the Tubb3enh:EGFP reporter, the control H2B-RFP vector, and control or Smad1/5 shRNA (sh-S1/5). (C) Quantification of luciferase activity (expressed in relative luciferase units [rlu]) driven by the pNeuroD reporter at 24 hpe with the Smad1/5 shRNA (sh-S1 or sh-S5) or control vectors. (D) Representative transverse sections of a chick neural tube at 48 hpe with control or Smad1/5 shRNA (sh-S1/5). DAPI, HuC/D, and H2B-RFP stain nuclei, differentiating neurons and electroporated cells. (E) Analysis of the ratios of the areas occupied by the VZ (Sox2 + ) and MZ (HuC/D + ) measured for the electroporated side and standardized to their contralateral controls. EP, electroporation. Error bars show means ± SEM. *, P

    Journal: The Journal of Cell Biology

    Article Title: The strength of SMAD1/5 activity determines the mode of stem cell division in the developing spinal cord

    doi: 10.1083/jcb.201307031

    Figure Lengend Snippet: SMAD1/5 inhibition triggers premature differentiation in a cell-autonomous manner. (A) Proportions of electroporated cells differentiated into neurons (H2B-RFP + ;HuC/D + ) obtained after electroporation of HH14 embryos with control (Ctrl) and Smad1/5 shRNA (sh-S1 and sh-S5) constructs, in sections of neural tubes recovered at the times indicated (hpe). (B) Representative sections stained for HuC/D expression 24 h after coelectroporation with the Tubb3enh:EGFP reporter, the control H2B-RFP vector, and control or Smad1/5 shRNA (sh-S1/5). (C) Quantification of luciferase activity (expressed in relative luciferase units [rlu]) driven by the pNeuroD reporter at 24 hpe with the Smad1/5 shRNA (sh-S1 or sh-S5) or control vectors. (D) Representative transverse sections of a chick neural tube at 48 hpe with control or Smad1/5 shRNA (sh-S1/5). DAPI, HuC/D, and H2B-RFP stain nuclei, differentiating neurons and electroporated cells. (E) Analysis of the ratios of the areas occupied by the VZ (Sox2 + ) and MZ (HuC/D + ) measured for the electroporated side and standardized to their contralateral controls. EP, electroporation. Error bars show means ± SEM. *, P

    Article Snippet: HH14 embryos electroporated with either pSox2:EGFP and a control H2B-RFP vector, pTis21:RFP and a control H2B-GFP vector, or pSox2:EGFP and pTis21:RFP were recovered at 24 hpe and processed for immunohistochemistry using the phospho-SMAD1/5/8 antibody (pS158; Cell Signaling Technology).

    Techniques: Inhibition, Electroporation, shRNA, Construct, Staining, Expressing, Plasmid Preparation, Luciferase, Activity Assay

    SMAD1/5 activity is required to maintain self-expanding divisions. (A–D) Analysis of neurogenic (PN + NN) and progenitor-generating (PP + PN) divisions in vivo. Transverse sections were stained for pH3 to identify mitotic progenitors at 24 hpe of HH14 embryos with control (Ctrl) or Smad1/5 shRNA (sh-S1 and sh-S5) constructs, together with the pTis21:RFP reporter and a control H2B-GFP vector (A) or with the pSox2:GFP reporter and control H2B-RFP vector (C). The higher magnification pictures originate from the corresponding insets. Proportions of mitotic electroporated (H2B-GFP + ;pH3 + [B] or H2B-RFP + ;pH3 + [D]) progenitors based on the activity of the pTis21:RFP (B) or pSox2:EGFP (D) reporters. (E) Proportions of the three modes of divisions (PP, PN, and NN) obtained 24 hpe with control or Smad1/5 shRNA vectors (sh-S1 or sh-S5). These percentages were deduced from the earlier results, considering that the %PP = %pTis21 − , %NN = %pSox2 − , and %PN = 100 − (%PP + %NN). (F) Proportions of the modes of divisions obtained 24 hpe with 0, 0.5, 1, or 2 µg/µl Somitabun. (G) Illustration of the increase in NN divisions obtained at the expense of PP divisions after SMAD1/5 inhibition in spinal neural progenitors. EP, electroporation; wt, wild type. Error bars show means ± SEM. *, P

    Journal: The Journal of Cell Biology

    Article Title: The strength of SMAD1/5 activity determines the mode of stem cell division in the developing spinal cord

    doi: 10.1083/jcb.201307031

    Figure Lengend Snippet: SMAD1/5 activity is required to maintain self-expanding divisions. (A–D) Analysis of neurogenic (PN + NN) and progenitor-generating (PP + PN) divisions in vivo. Transverse sections were stained for pH3 to identify mitotic progenitors at 24 hpe of HH14 embryos with control (Ctrl) or Smad1/5 shRNA (sh-S1 and sh-S5) constructs, together with the pTis21:RFP reporter and a control H2B-GFP vector (A) or with the pSox2:GFP reporter and control H2B-RFP vector (C). The higher magnification pictures originate from the corresponding insets. Proportions of mitotic electroporated (H2B-GFP + ;pH3 + [B] or H2B-RFP + ;pH3 + [D]) progenitors based on the activity of the pTis21:RFP (B) or pSox2:EGFP (D) reporters. (E) Proportions of the three modes of divisions (PP, PN, and NN) obtained 24 hpe with control or Smad1/5 shRNA vectors (sh-S1 or sh-S5). These percentages were deduced from the earlier results, considering that the %PP = %pTis21 − , %NN = %pSox2 − , and %PN = 100 − (%PP + %NN). (F) Proportions of the modes of divisions obtained 24 hpe with 0, 0.5, 1, or 2 µg/µl Somitabun. (G) Illustration of the increase in NN divisions obtained at the expense of PP divisions after SMAD1/5 inhibition in spinal neural progenitors. EP, electroporation; wt, wild type. Error bars show means ± SEM. *, P

    Article Snippet: HH14 embryos electroporated with either pSox2:EGFP and a control H2B-RFP vector, pTis21:RFP and a control H2B-GFP vector, or pSox2:EGFP and pTis21:RFP were recovered at 24 hpe and processed for immunohistochemistry using the phospho-SMAD1/5/8 antibody (pS158; Cell Signaling Technology).

    Techniques: Activity Assay, In Vivo, Staining, shRNA, Construct, Plasmid Preparation, Inhibition, Electroporation

    Strong SMAD1/5 activity promotes PP divisions at the expense of PN and NN divisions. (A and B) Proportions of mitotic electroporated progenitors (H2B-GFP + ;pH3 + [A] or H2B-RFP + ;pH3 + [B]) based on the activity of the pTis21:RFP (A) or pSox2:EGFP (B) reporters at 24 h after coelectroporation of HH14 embryos with control (Ctrl), SMAD1-SD (1-SD), or SMAD5-SD (5-SD). (C) Percentages of the three modes of divisions (PP, PN, and NN) obtained at 24 hpe with the constructs indicated and deduced from earlier results, considering that %PP = %pTis21 − , %NN = %pSox2 − , and %PN = 100 − (%PP + %NN). (D) Illustration of the increase in PP divisions obtained at the expense of PN and NN divisions after SMAD1/5 overactivation in spinal progenitors. EP, electroporation; wt, wild type. Error bars show means ± SEM. *, P

    Journal: The Journal of Cell Biology

    Article Title: The strength of SMAD1/5 activity determines the mode of stem cell division in the developing spinal cord

    doi: 10.1083/jcb.201307031

    Figure Lengend Snippet: Strong SMAD1/5 activity promotes PP divisions at the expense of PN and NN divisions. (A and B) Proportions of mitotic electroporated progenitors (H2B-GFP + ;pH3 + [A] or H2B-RFP + ;pH3 + [B]) based on the activity of the pTis21:RFP (A) or pSox2:EGFP (B) reporters at 24 h after coelectroporation of HH14 embryos with control (Ctrl), SMAD1-SD (1-SD), or SMAD5-SD (5-SD). (C) Percentages of the three modes of divisions (PP, PN, and NN) obtained at 24 hpe with the constructs indicated and deduced from earlier results, considering that %PP = %pTis21 − , %NN = %pSox2 − , and %PN = 100 − (%PP + %NN). (D) Illustration of the increase in PP divisions obtained at the expense of PN and NN divisions after SMAD1/5 overactivation in spinal progenitors. EP, electroporation; wt, wild type. Error bars show means ± SEM. *, P

    Article Snippet: HH14 embryos electroporated with either pSox2:EGFP and a control H2B-RFP vector, pTis21:RFP and a control H2B-GFP vector, or pSox2:EGFP and pTis21:RFP were recovered at 24 hpe and processed for immunohistochemistry using the phospho-SMAD1/5/8 antibody (pS158; Cell Signaling Technology).

    Techniques: Activity Assay, Construct, Electroporation

    PP, PN, and NN divisions co-occur during spinal cord neurogenesis. (A) In ovo electroporation (EP) of the pTis21:RFP and pSox2:GFP reporters allows us to identify and discriminate the populations and divisions of PP, PN, and NN progenitors within the developing chick spinal cord. (B) Representative neural tube section obtained at 24 hpe of HH14 embryos, showing GFP + ;RFP − (PP), GFP + ;RFP + (PN), and GFP - ;RFP + (NN) cells in response to differential activities of the pTis21:RFP and pSox2:GFP reporters. The inset shows the neural tube morphology, with nuclei stained with DAPI. (C) The proportions of PP, PN, and NN divisions were assessed at 16 hpe at different developmental points, with a combination of the pSox2:EGFP and pTis21:RFP reporters and pH3 staining to reveal mitoses. Error bars show means ± SEM. (D) Illustration of the three modes of divisions occurring along the dorsal–ventral axis of a developing spinal cord during interneuron neurogenesis. (E–G) Representative neural tube sections obtained 24 h after coelectroporation of HH14 embryos with combinations of the TOP:H2B-RFP (E), GBS:H2B-RFP (F), or BRE:EGFP (G) with their respective controls. The insets show the neural tube morphology, with nuclei stained with DAPI. The right images show the signal observed in response to the specific activity of the corresponding reporter. Bars, 50 µM.

    Journal: The Journal of Cell Biology

    Article Title: The strength of SMAD1/5 activity determines the mode of stem cell division in the developing spinal cord

    doi: 10.1083/jcb.201307031

    Figure Lengend Snippet: PP, PN, and NN divisions co-occur during spinal cord neurogenesis. (A) In ovo electroporation (EP) of the pTis21:RFP and pSox2:GFP reporters allows us to identify and discriminate the populations and divisions of PP, PN, and NN progenitors within the developing chick spinal cord. (B) Representative neural tube section obtained at 24 hpe of HH14 embryos, showing GFP + ;RFP − (PP), GFP + ;RFP + (PN), and GFP - ;RFP + (NN) cells in response to differential activities of the pTis21:RFP and pSox2:GFP reporters. The inset shows the neural tube morphology, with nuclei stained with DAPI. (C) The proportions of PP, PN, and NN divisions were assessed at 16 hpe at different developmental points, with a combination of the pSox2:EGFP and pTis21:RFP reporters and pH3 staining to reveal mitoses. Error bars show means ± SEM. (D) Illustration of the three modes of divisions occurring along the dorsal–ventral axis of a developing spinal cord during interneuron neurogenesis. (E–G) Representative neural tube sections obtained 24 h after coelectroporation of HH14 embryos with combinations of the TOP:H2B-RFP (E), GBS:H2B-RFP (F), or BRE:EGFP (G) with their respective controls. The insets show the neural tube morphology, with nuclei stained with DAPI. The right images show the signal observed in response to the specific activity of the corresponding reporter. Bars, 50 µM.

    Article Snippet: HH14 embryos electroporated with either pSox2:EGFP and a control H2B-RFP vector, pTis21:RFP and a control H2B-GFP vector, or pSox2:EGFP and pTis21:RFP were recovered at 24 hpe and processed for immunohistochemistry using the phospho-SMAD1/5/8 antibody (pS158; Cell Signaling Technology).

    Techniques: In Ovo, Electroporation, Staining, Activity Assay

    Loss of SMAD1/5 activity causes a specific shortening of the S phase. (A) Representative sections of neural tubes at 48 hpe, after incorporation of BrdU in the last 1 and 12 h. BrdU staining was observed in a fraction of the electroporated H2B-RFP + neural progenitors that were still present in the VZ (dashed lines). (B) BrdU labeling index calculated for neural progenitors electroporated (H2B-RFP + ) with control (Ctrl), Smad1 (sh-S1), or Smad5 (sh-S5) shRNA vectors. Dashed lines show the growth fraction (y axis) and the duration of BrdU incorporation needed to reach the plateau, which represents the (Tc − T S ) value ( x axis). (C) Representative sections showing BrdU staining in a fraction of the mitotic (pH3 + ) electroporated (GFP + ) neural progenitors. (D) Mitotic BrdU labeling index calculated for cells electroporated (GFP + ) with control, Smad1 , or Smad5 (sh-S5) shRNA vectors. Dashed lines show the duration of the BrdU incorporation needed to label 50% of the cell population ( y axis), which represents the mean T G2 value ( x axis). (E) Mitotic index calculated for cells electroporated (GFP + ) with control, Smad1 (sh-S1), or Smad5 (sh-S5) shRNA vectors. Both the mean values obtained from counting and those corrected with respect to differential growth fraction values are given. (F) Graphical representation of the duration of the cell cycle (Tc) and its distinct phases measured for control, Smad1 (sh-S1), or Smad5 shRNA (sh-S5) electroporated cells. (G–K) Illustration of the method used to assess neural progenitor divisions in vivo (G), after loss (H and I) or gain of function (J and K) of SMAD1/5 activity. (H and J) Representative pictures of the CellTrace violet intensity measured by flow cytometry in dissociated cells at 48 hpe with the indicated constructs. The blue bars highlight the mean intensity obtained in the control condition. (I and K) Quantification of the mean CellTrace violet intensity measured at 48 hpe with the indicated constructs. a.u., arbitrary unit; EP, electroporation. Error bars show means ± SEM. **, P

    Journal: The Journal of Cell Biology

    Article Title: The strength of SMAD1/5 activity determines the mode of stem cell division in the developing spinal cord

    doi: 10.1083/jcb.201307031

    Figure Lengend Snippet: Loss of SMAD1/5 activity causes a specific shortening of the S phase. (A) Representative sections of neural tubes at 48 hpe, after incorporation of BrdU in the last 1 and 12 h. BrdU staining was observed in a fraction of the electroporated H2B-RFP + neural progenitors that were still present in the VZ (dashed lines). (B) BrdU labeling index calculated for neural progenitors electroporated (H2B-RFP + ) with control (Ctrl), Smad1 (sh-S1), or Smad5 (sh-S5) shRNA vectors. Dashed lines show the growth fraction (y axis) and the duration of BrdU incorporation needed to reach the plateau, which represents the (Tc − T S ) value ( x axis). (C) Representative sections showing BrdU staining in a fraction of the mitotic (pH3 + ) electroporated (GFP + ) neural progenitors. (D) Mitotic BrdU labeling index calculated for cells electroporated (GFP + ) with control, Smad1 , or Smad5 (sh-S5) shRNA vectors. Dashed lines show the duration of the BrdU incorporation needed to label 50% of the cell population ( y axis), which represents the mean T G2 value ( x axis). (E) Mitotic index calculated for cells electroporated (GFP + ) with control, Smad1 (sh-S1), or Smad5 (sh-S5) shRNA vectors. Both the mean values obtained from counting and those corrected with respect to differential growth fraction values are given. (F) Graphical representation of the duration of the cell cycle (Tc) and its distinct phases measured for control, Smad1 (sh-S1), or Smad5 shRNA (sh-S5) electroporated cells. (G–K) Illustration of the method used to assess neural progenitor divisions in vivo (G), after loss (H and I) or gain of function (J and K) of SMAD1/5 activity. (H and J) Representative pictures of the CellTrace violet intensity measured by flow cytometry in dissociated cells at 48 hpe with the indicated constructs. The blue bars highlight the mean intensity obtained in the control condition. (I and K) Quantification of the mean CellTrace violet intensity measured at 48 hpe with the indicated constructs. a.u., arbitrary unit; EP, electroporation. Error bars show means ± SEM. **, P

    Article Snippet: HH14 embryos electroporated with either pSox2:EGFP and a control H2B-RFP vector, pTis21:RFP and a control H2B-GFP vector, or pSox2:EGFP and pTis21:RFP were recovered at 24 hpe and processed for immunohistochemistry using the phospho-SMAD1/5/8 antibody (pS158; Cell Signaling Technology).

    Techniques: Activity Assay, BrdU Staining, Labeling, shRNA, BrdU Incorporation Assay, In Vivo, Flow Cytometry, Cytometry, Construct, Electroporation

    Integrin α6β4 signaling promotes recruitment of p53 and 53BP1 to chromatin in response to cisplatin treatment. BT549 cells (EV and β4) plated on laminin-1 and treated with 10 μM cisplatin for 24 hrs were harvested for cytosolic or nuclear fractions and immuno-blotted for phospho-p53 using actin and p84 as controls (A) or for immunocytochemistry staining for phospho-p53 S15 and phospho-53BP1 S1778 as indicated (B, scale bars, 20µm). (C) Subcellular protein fractionation was performed on cells treated as in (A) and noted fractions were immunoblotted with DDR proteins as indicated. Tubulin was used as the marker for total and cytosolic protein, Histone H2B and p84 were used as the markers for nuclear fractions.

    Journal: bioRxiv

    Article Title: Integrin α6β4 signaling switches DNA repair from homologous recombination to non-homologous end-joining pathway to sensitize breast cancer cells to cisplatin

    doi: 10.1101/785873

    Figure Lengend Snippet: Integrin α6β4 signaling promotes recruitment of p53 and 53BP1 to chromatin in response to cisplatin treatment. BT549 cells (EV and β4) plated on laminin-1 and treated with 10 μM cisplatin for 24 hrs were harvested for cytosolic or nuclear fractions and immuno-blotted for phospho-p53 using actin and p84 as controls (A) or for immunocytochemistry staining for phospho-p53 S15 and phospho-53BP1 S1778 as indicated (B, scale bars, 20µm). (C) Subcellular protein fractionation was performed on cells treated as in (A) and noted fractions were immunoblotted with DDR proteins as indicated. Tubulin was used as the marker for total and cytosolic protein, Histone H2B and p84 were used as the markers for nuclear fractions.

    Article Snippet: For total cell lysates, treated cells were harvested in lysis buffer with phosphatase inhibitors (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 µg/ml leupeptin, 1 mM PMSF), sonicated and total cell lysates were subjected to SDS-PAGE, transferred to PVDF membrane and immunoblotted with various antibodies (Cell Signaling Technology). β-actin (monoclonal antibody; Sigma) was used as a loading control for total lysates, and tubulin (Millipore-Sigma) for cytosolic, p84 (GeneTex) for nuclear and histone H2B (Cell Signaling Technology) for chromatin bound fractions.

    Techniques: Immunocytochemistry, Staining, Fractionation, Marker

    Demonstration of antibody specificity to D-isoAsp-H2B (A) Western blots of histones from pcmt1 WT or KO mouse brain using a commercial pan-H2B antibody (H2B, top row), and our custom antibody made against the D-isoAsp-H2B synthetic peptide (H2B*, bottom row). Because the same histone preps were used for both blots, the top row serves as a loading control for the bottom row. (B) There is little difference (p = 0.235) between the WT and KO brain extracts when the pan-H2B antibody is used for blotting, but a large and significant (p = 0.003) difference when the H2B* antibody is used. Panel C shows that, when normalized to the pan-H2B signals, immuno-reactivity with the H2B* antibody is 2X stronger (p = 0.001) in the WT vs. KO extracts.

    Journal: Amino acids

    Article Title: The D-isoAsp-25 variant of histone H2B is highly enriched in active chromatin: potential role in the regulation of gene expression?

    doi: 10.1007/s00726-015-2140-9

    Figure Lengend Snippet: Demonstration of antibody specificity to D-isoAsp-H2B (A) Western blots of histones from pcmt1 WT or KO mouse brain using a commercial pan-H2B antibody (H2B, top row), and our custom antibody made against the D-isoAsp-H2B synthetic peptide (H2B*, bottom row). Because the same histone preps were used for both blots, the top row serves as a loading control for the bottom row. (B) There is little difference (p = 0.235) between the WT and KO brain extracts when the pan-H2B antibody is used for blotting, but a large and significant (p = 0.003) difference when the H2B* antibody is used. Panel C shows that, when normalized to the pan-H2B signals, immuno-reactivity with the H2B* antibody is 2X stronger (p = 0.001) in the WT vs. KO extracts.

    Article Snippet: Finally, each immunoprecipitation was subjected to Western analysis with pan-H3, pan-H2B, or the H2B* antibody ( ).

    Techniques: Western Blot

    D-isoAsp-H2B is enriched in active chromatin Chromatin was prepared from all 6 WT mouse brains (WT1-3 males, WT4–6 females) and each preparation was subjected to immunoprecipitations with antibody to acetylated histone H3 (H3K9; Ac) and with methylated histone H3 (H3K9Me3; Me). (A) The immunoprecipitates were than subjected to Western blotting using a pan-H3 antibody (H3), a pan-H2B antibody (H2B), and our D-isoAsp-H2B antibody (H2B*). (B) The Ac/Me band ratios are the same, as expected, when immunoblotting for pan-H3 or pan-H2B, whereas the ratio when using H2B* is about 55% higher.

    Journal: Amino acids

    Article Title: The D-isoAsp-25 variant of histone H2B is highly enriched in active chromatin: potential role in the regulation of gene expression?

    doi: 10.1007/s00726-015-2140-9

    Figure Lengend Snippet: D-isoAsp-H2B is enriched in active chromatin Chromatin was prepared from all 6 WT mouse brains (WT1-3 males, WT4–6 females) and each preparation was subjected to immunoprecipitations with antibody to acetylated histone H3 (H3K9; Ac) and with methylated histone H3 (H3K9Me3; Me). (A) The immunoprecipitates were than subjected to Western blotting using a pan-H3 antibody (H3), a pan-H2B antibody (H2B), and our D-isoAsp-H2B antibody (H2B*). (B) The Ac/Me band ratios are the same, as expected, when immunoblotting for pan-H3 or pan-H2B, whereas the ratio when using H2B* is about 55% higher.

    Article Snippet: Finally, each immunoprecipitation was subjected to Western analysis with pan-H3, pan-H2B, or the H2B* antibody ( ).

    Techniques: Methylation, Western Blot

    (A) Western blot images created by Compass Software (ProteinSimple, Santa Clara, CA, USA) representing modified and total histone expression in the prefrontal cortex (PFC) for each treatment group. No differences in total histone expression was observed for any histone protein. (B) Levels of acetyl-H2b (AH2b), acetyl-H3 (AH3), and acetyl-H4 (AH4) were significantly decreased compared to sham. No differences were observed between injury groups. *Indicates p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Astrocyte Reactivity Following Blast Exposure Involves Aberrant Histone Acetylation

    doi: 10.3389/fnmol.2016.00064

    Figure Lengend Snippet: (A) Western blot images created by Compass Software (ProteinSimple, Santa Clara, CA, USA) representing modified and total histone expression in the prefrontal cortex (PFC) for each treatment group. No differences in total histone expression was observed for any histone protein. (B) Levels of acetyl-H2b (AH2b), acetyl-H3 (AH3), and acetyl-H4 (AH4) were significantly decreased compared to sham. No differences were observed between injury groups. *Indicates p

    Article Snippet: Specific antibodies for glial fibrillary acidic protein (GFAP; Abcam, Cambridge, UK), Ionized Calcium-Binding Adapater Molecule 1 (IBA-1; Biocare Medical, Concord, CA, USA), H2a (Cell Signaling Technologies, Danvers, MA, USA), H2b (Cell Signaling Technologies, Danvers, MA, USA), H3 (Cell Signaling Technologies, Danvers, MA, USA), H4 (Cell Signaling Technologies, Danvers, MA, USA), acetyl-H2a (AH2a; Cell Signaling Technologies, Danvers, MA, USA), acetyl-H2b (AH2b; Cell Signaling Technologies, Danvers, MA, USA), acetyl-H3 (AH3; Cell Signaling Technologies, Danvers, MA, USA), and acetyl-H4 (AH4; Cell Signaling Technologies, Danvers, MA, USA) were used.

    Techniques: Western Blot, Software, Modification, Expressing