histone h4 Search Results


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  • 99
    Millipore histone h 4
    Apoptotic cells exhibit reduced staining for acetylated histone H4. Immunofluorescent double labeling of cells in the ganglion cell layer of a DBA/2J mouse. Frozen sections were double labeled with antibodies against the histone variant γH2AX
    Histone H 4, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam histone h3
    Partially closed chromatin structure and increased H3K methylation in the ATM promoter in Vav-IDH1-KI cells (A) ATAC-qPCR to analyze the indicated genes in LSK cells from Vav-IDH1-KI and WT mice (n=3). Data are the mean ± SD. (B) Immunoblot to detect the indicated methylated forms of <t>histone</t> H3 in Lin − cells from Vav-IDH1-KI and WT mice. (C) ChIP-qPCR analysis of the Atm promoter in LSK cells from Vav-IDH1-KI and WT mice (n=3) using anti-histone H3-K9me3 Ab. Data are the mean ± SD. (D) Immunoblot to detect the indicated proteins in LSK cells isolated from Vav-IDH1-KI and WT mice and cultured with/without UNC1999 (1 µM) or UNC0642(0.25 or 1 µM). UT, untreated. (E) qRT-PCR determination of Atm .
    Histone H3, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 6614 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    ABclonal histones
    Partially closed chromatin structure and increased H3K methylation in the ATM promoter in Vav-IDH1-KI cells (A) ATAC-qPCR to analyze the indicated genes in LSK cells from Vav-IDH1-KI and WT mice (n=3). Data are the mean ± SD. (B) Immunoblot to detect the indicated methylated forms of <t>histone</t> H3 in Lin − cells from Vav-IDH1-KI and WT mice. (C) ChIP-qPCR analysis of the Atm promoter in LSK cells from Vav-IDH1-KI and WT mice (n=3) using anti-histone H3-K9me3 Ab. Data are the mean ± SD. (D) Immunoblot to detect the indicated proteins in LSK cells isolated from Vav-IDH1-KI and WT mice and cultured with/without UNC1999 (1 µM) or UNC0642(0.25 or 1 µM). UT, untreated. (E) qRT-PCR determination of Atm .
    Histones, supplied by ABclonal, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Signaling Technology Inc histone h3
    Proximity biotin ligation assay of KIF2A isoforms in Neuro2A cells. A . Immunofluorescence staining of myc (red), Streptavidin (green) and DNA (blue) in Neuro2A cells transfected with myc BirA* , myc BirA*-Kif2a.1, mycBirA*-Kif2a.2, mycBirA*-Kif2a.3  or  mycBirA*-Kif2a H321D  separately. Transfected cells were identified based on myc immunostaining. Scale bar, 10 µm.  B . Top panel: Immunoblotting with anti-myc showing expression of mycBirA*-KIF2A in Neuro2A cells. Middle panel: Immunoblotting with anti-biotin confirming biotinylation. Bottom panel: Anti-Histone H3 as loading control.  C . Pull-down with streptavidin beads followed by anti-biotin immunoblotting of Neuro2A cell lysates. Biotin supplementation to cell media is indicated.
    Histone H3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 5209 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam abcam histone h3
    Proximity biotin ligation assay of KIF2A isoforms in Neuro2A cells. A . Immunofluorescence staining of myc (red), Streptavidin (green) and DNA (blue) in Neuro2A cells transfected with myc BirA* , myc BirA*-Kif2a.1, mycBirA*-Kif2a.2, mycBirA*-Kif2a.3  or  mycBirA*-Kif2a H321D  separately. Transfected cells were identified based on myc immunostaining. Scale bar, 10 µm.  B . Top panel: Immunoblotting with anti-myc showing expression of mycBirA*-KIF2A in Neuro2A cells. Middle panel: Immunoblotting with anti-biotin confirming biotinylation. Bottom panel: Anti-Histone H3 as loading control.  C . Pull-down with streptavidin beads followed by anti-biotin immunoblotting of Neuro2A cell lysates. Biotin supplementation to cell media is indicated.
    Abcam Histone H3, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Cell Signaling Technology Inc anti histone h4
    The effect of oncoVV-HddSBL on <t>histone</t> modification. C6 glioblastoma cells were treated with PBS, 5 MOI of oncoVV or oncoVV-HddSBL, and histone H3, H4, H3R8, and H4R3 asymmetric dimethylation levels, as well as the expression of FLAG-tagged HddSBL were analyzed by Western blot.
    Anti Histone H4, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 177 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology histone h3
    TGF-β-associated gene expression profiles induced by treatment with mitogen-activated protein kinase inhibitors. (A) CRL-2097 human dermal fibroblasts were cultured for 15-60 min in the presence or absence of 10 µ M U0126 (ERK i ) or 10 µ M SP600125 (JNK i ) and subjected to western blot analysis for p-ERK1/2. <t>Histone</t> H3 was used as a loading control. Fibroblasts incubated with recombinant human FGF2 for 30 min were used as a positive control for p-ERK1/2 expression. (B) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ) or 10 µ M SB202190 (p38 i ) until day 4. Expression levels of TGF-β-associated transcripts TGFB1 , TGFB2 , TGFBR1 , TGFBR2 and TGFBR3 were determined relative to fibroblasts cultured under control conditions by reverse transcription-quantitative polymerase chain reaction. GAPDH expression was used as an internal control. Expression levels per transcript were compared using an one-way analysis of variance and post-hoc Holm-Sidak analysis. * P
    Histone H3, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1550 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Active Motif histone h3
    <t>Histone</t> acetylation and E2F1 binding at the BRCA1 and RAD51 promoter regions. ( A, B ) CBP and p300 recruitment to the BRCA1 and RAD51 promoter regions. H1299 cells were subjected to ChIP using either control IgG or antibodies against CBP and p300. Immunoprecipitated DNAs were subjected to quantitative PCR for the detection of the promoter region of BRCA1 (A) or RAD51 (B) and for the detection of the non-promoter region of GAPDH . Normalized ChIP enrichment was calculated to show the degree of enrichment following pulldown with anti-p300 and anti-CBP antibodies. First, ChIP enrichment at the test sites ( BRCA1 or RAD51 promoter and the GAPDH gene body) was calculated as the fraction of total input DNA that was pulled down by the specific antibody or the non-specific control IgG. Then, normalized ChIP enrichment at the test sites was calculated by dividing the ChIP enrichment of the specific antibody by that of the non-specific control IgG. Data represent mean values ± SD. ( C–J ) Impaired histone acetylation and changes in E2F1 and E2F4 binding at BRCA1 and RAD51 promoter regions caused by knockdown of CBP and p300. H1299 cells pre-transfected with non-targeting (siNT) or CBP and p300 (siC+p) siRNAs were subjected to ChIP assays. DNA immunoprecipitated with anti-acetylated H3 K18 (H3 AceK18) (C, D), anti-acetylated H4 (H4 AceK5/8/12/16) (E, F), anti-E2F1 (G, H), or anti-E2F4 (I, J) antibodies was subjected to quantitative PCR to detect the promoter regions of BRCA1 (C, E, G, I) and RAD51 (D, F, H, J), and the gene body region of GAPDH . Relative ChIP enrichment was calculated to show the decrease/increase in enrichment at these sites following gene knockdown. The relative ChIP enrichment was calculated by dividing the ChIP enrichment at the BRCA1 or RAD51 promoter and the GAPDH gene body region in the siRNA-treated sample by the ChIP enrichment at the BRCA1 or RAD51 promoter in the siNT-treated sample. Data represent the mean ± SD.
    Histone H3, supplied by Active Motif, used in various techniques. Bioz Stars score: 92/100, based on 323 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Upstate Biotechnology Inc histone h4
    Recovery of histone acetylation by overexpression of wt p300 HAT. HS27/vIRF cells were transfected with wt p300 (lane 3) or p300 ΔHAT mutant (lane 4). HS27/cDNA3 (lane 1) and HS27/vIRF (lane 2) were included as controls. Identical amounts of proteins from cell lysates were used for immunoblotting analysis with an antibody specific for the acetylated histone H4. The bottom panel shows the amount of cellular <t>histone</t> H4 protein in each lane, detected by an anti-H4 antibody. Arrows indicate the acetylated form of histone H4 (Ac-H4) or total histone H4 (H4). Numbers at left are molecular masses in kilodaltons.
    Histone H4, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 92/100, based on 621 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GeneTex histone h4
    GO/CDDP triggered direct association of histones H1/H4 and CDDP with LC3. (A) SDS-PAGE analysis of LC3-immunocomplex from the lysate (Lys) and nuclear (Nuc) fractions of cells with or without GO/CDDP treatment. (B) Western blot analysis of proteins (~11 kDa and ~35 kDa) derived from the nuclear LC3-immunocomplex. (C) Immunofluorescence microscopy of cells with or without GO/CDDP treatment. The cells were immunolabeled with primary antibody for histone H1 or H4 and counter stained with DAPI. (D) Percentages of cells positive with total histone H1, phosphorylated histone H1 (p-histone H1), <t>histone</t> H4 and acetylated histone H4 (H4K16ac). (E) Amount of CDDP associated with nuclear LC3 immunocomplex. The nuclear LC3 immunocomplex was analyzed by ICP-MS to quantify the amount of platinum (Pt) in CDDP. The data represent mean ± s.d of at least 3 independent culture experiments.
    Histone H4, supplied by GeneTex, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Abcam histone 4
    GO/CDDP triggered direct association of histones H1/H4 and CDDP with LC3. (A) SDS-PAGE analysis of LC3-immunocomplex from the lysate (Lys) and nuclear (Nuc) fractions of cells with or without GO/CDDP treatment. (B) Western blot analysis of proteins (~11 kDa and ~35 kDa) derived from the nuclear LC3-immunocomplex. (C) Immunofluorescence microscopy of cells with or without GO/CDDP treatment. The cells were immunolabeled with primary antibody for histone H1 or H4 and counter stained with DAPI. (D) Percentages of cells positive with total histone H1, phosphorylated histone H1 (p-histone H1), <t>histone</t> H4 and acetylated histone H4 (H4K16ac). (E) Amount of CDDP associated with nuclear LC3 immunocomplex. The nuclear LC3 immunocomplex was analyzed by ICP-MS to quantify the amount of platinum (Pt) in CDDP. The data represent mean ± s.d of at least 3 independent culture experiments.
    Histone 4, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche histone h1
    Primer extension activity of RT HIV-1 using the 17mer/44mer primer/template duplex. The experiments were conducted for the times indicated in the figure (5–90 min) using undamaged templates (lanes 2–5), the template containing single, site-specific 1,2-GG intrastrand CL of cisplatin (lanes 6–9) and the template containing single DPCL formed by the transformation of the template containing site-specific 1,2-GG intrastrand CL of cisplatin incubated with <t>histone</t> H1 (lanes 10–13). Lane 1, 17-mer primer. The pause sites opposite the platinated guanines and the nucleotide preceding the platinated guanines (thymine residue on the 3′ side of the CL) are marked 34, 33, 32, respectively. The nucleotide sequences of the templates and the primers are shown beneath the gels. See the text for other details.
    Histone H1, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 835 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    BPS Bioscience histone h4
    Cytotoxic effects of extracellular <t>histones</t> on alveolar epithelial cells. A ) LDH release from LA-4 and MLE-12 lung alveolar epithelial cells exposed to purified histones (50 μg/ml, 1 h), chromogenic assay. B ) [Ca 2+ ] i staining in untreated (Ctrl)
    Histone H4, supplied by BPS Bioscience, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GenScript histone h4
    CARM1 represses γ-globin expression. A , Western blot analysis of indicated protein levels in cell lysates from the scramble control (Scr), CARM1-KD1, and CARM1-KD2 Lys-562 cells. GAPDH and <t>histone</t> H4 were used as loading controls. Blots are representative of three independent experiments. B and C , quantitative real-time PCR analysis of CARM1 ( B ) and γ-globin ( C ) mRNA level in Scr, CARM1-KD1, and CARM1-KD2 Lys-562 cells normalized to β-actin mRNA. The results are shown as the mean ± S.D. from three independent experiments. Two-tailed Student's t -tests were used to compare means. **, p
    Histone H4, supplied by GenScript, used in various techniques. Bioz Stars score: 92/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boehringer Mannheim histone h1
    Cross-linking of E7 peptides to CDK2/cyclin A complex. (A) HPV16 E7 (1.8 μM) and E7-derived peptides (50 μM) stimulate CDK2/cyclin A histone kinase activity. (B) Benzo-CYS-9-48-bio and benzo-NH 2 -9-48-bio peptides associate with cyclin A/CDK2 complex following UV irradiation. <t>Histone</t> H1 is not cross-linked under identical conditions. (C) CDK2/cyclin A (0.1 μM) and benzo-CYS-9-48-bio (0.5 μM) were with or without competitor E7 or E7 peptide. Both full-length 16E7 and the 9-48 16E7 peptide compete with the benzo-CYS-9-48-bio peptide for binding to the cyclin A/CDK2 complex. These results suggest that labeling of the CDK2/cyclin A complex by benzo-CYS-9-48-bio is specific and is not due to indiscriminate labeling of the complex. (D) Immunoprecipitation (IP) followed by Western blotting for incorporation of biotin-containing peptide into immunoprecipitated target protein(s) demonstrates that biotin-reactive bands in Western blots are cyclin A and CDK2 and that reactivity of these bands with benzo-CYS-9-48-bio peptide is dependent upon UV. (E) Cross-linking of benzophenone-containing peptides to increasing amounts of GST, cyclin A, or CDK2 demonstrates a strong preference of the peptide for cyclin A.
    Histone H1, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 436 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Merck KGaA histone h3
    An ACLY knockdown does not recapitulate the phenotype of ACLY inhibitor-treated MDMs. (A–D) Q-PCR analysis of mRNA expression of ALOX15, CCL17, F13A1, and ACLY in MDMs transfected with 50 nM ACLY siRNA for 96 h prior to 24-h treatment with 20 ng/mL IL-4. (E–G) Western blot analysis of ACLY protein expression (E) , assay of enzymatic activity (F) , and Western blot analysis of <t>histone</t> H3 acetylation at K27 and K14 (G) 96 h post-transfection with ACLY siRNA. ** p
    Histone H3, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biomol GmbH histone h4
    Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.
    Histone H4, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc anti acetyl histone h3 antibody
    PITX2 binds to  SLC13A3  upstream elements. ChIP was used as template for standard PCR. Antibodies against PITX2 and acetyl-histone H3 (Lys9) (positive control for transcriptionally active chromatin) yielded template from which fragments of the SLC13A3
    Anti Acetyl Histone H3 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam histone h4 polyclonal antibody
    PITX2 binds to  SLC13A3  upstream elements. ChIP was used as template for standard PCR. Antibodies against PITX2 and acetyl-histone H3 (Lys9) (positive control for transcriptionally active chromatin) yielded template from which fragments of the SLC13A3
    Histone H4 Polyclonal Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Apoptotic cells exhibit reduced staining for acetylated histone H4. Immunofluorescent double labeling of cells in the ganglion cell layer of a DBA/2J mouse. Frozen sections were double labeled with antibodies against the histone variant γH2AX

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Silencing of Fem1cR3 Gene Expression in the DBA/2J Mouse Precedes Retinal Ganglion Cell Death and Is Associated with Histone Deacetylase Activity

    doi: 10.1167/iovs.11-8872

    Figure Lengend Snippet: Apoptotic cells exhibit reduced staining for acetylated histone H4. Immunofluorescent double labeling of cells in the ganglion cell layer of a DBA/2J mouse. Frozen sections were double labeled with antibodies against the histone variant γH2AX

    Article Snippet: Similarly, colocalization of acetylated histone H4 (AcH4) and γH2AX showed that normal-appearing (stage I) cells had robust AcH4 labeling ( ).

    Techniques: Staining, Labeling, Variant Assay

    HDAC3 concentrates in nuclei of apoptotic cells. Immunofluorescent double labeling of cells in the ganglion cell layer of a DBA/2J mouse. Frozen sections were double labeled with antibodies against the histone variant γH2AX to identify dying cells,

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Silencing of Fem1cR3 Gene Expression in the DBA/2J Mouse Precedes Retinal Ganglion Cell Death and Is Associated with Histone Deacetylase Activity

    doi: 10.1167/iovs.11-8872

    Figure Lengend Snippet: HDAC3 concentrates in nuclei of apoptotic cells. Immunofluorescent double labeling of cells in the ganglion cell layer of a DBA/2J mouse. Frozen sections were double labeled with antibodies against the histone variant γH2AX to identify dying cells,

    Article Snippet: Similarly, colocalization of acetylated histone H4 (AcH4) and γH2AX showed that normal-appearing (stage I) cells had robust AcH4 labeling ( ).

    Techniques: Labeling, Variant Assay

    (A) Representative low-magnification photomicrographs of acetylated histones H3-K9 or H4-K8 gold-immunolabeling in central nucleus of amygdala (CeA) of P and NP rats treated with either vehicle [P (Vehicle) and NP(Vehicle)] or TSA [P(TSA) and NP(TSA)]

    Journal: The international journal of neuropsychopharmacology / official scientific journal of the Collegium Internationale Neuropsychopharmacologicum (CINP)

    Article Title: Effects of histone deacetylase inhibitors on amygdaloid histone acetylation and neuropeptide Y expression: A role in anxiety-like and alcohol-drinking behaviors

    doi: 10.1017/S1461145714000054

    Figure Lengend Snippet: (A) Representative low-magnification photomicrographs of acetylated histones H3-K9 or H4-K8 gold-immunolabeling in central nucleus of amygdala (CeA) of P and NP rats treated with either vehicle [P (Vehicle) and NP(Vehicle)] or TSA [P(TSA) and NP(TSA)]

    Article Snippet: The gold-immunolabeling histochemical procedure described previously ( ; ; ) was employed using antibodies against HDAC2 (1:100 dilution, MBL International, Woburn, MA) and HDAC4 (1:100 dilution, MBL International), acetylated histone H3-K9 (1:500 dilution, Millipore, Billerica, MA), acetylated histone H4-K8 (1:500 dilution, Millipore) and NPY (1:500 dilution, Immunostar, Hudson, WI).

    Techniques: Immunolabeling

    Partially closed chromatin structure and increased H3K methylation in the ATM promoter in Vav-IDH1-KI cells (A) ATAC-qPCR to analyze the indicated genes in LSK cells from Vav-IDH1-KI and WT mice (n=3). Data are the mean ± SD. (B) Immunoblot to detect the indicated methylated forms of histone H3 in Lin − cells from Vav-IDH1-KI and WT mice. (C) ChIP-qPCR analysis of the Atm promoter in LSK cells from Vav-IDH1-KI and WT mice (n=3) using anti-histone H3-K9me3 Ab. Data are the mean ± SD. (D) Immunoblot to detect the indicated proteins in LSK cells isolated from Vav-IDH1-KI and WT mice and cultured with/without UNC1999 (1 µM) or UNC0642(0.25 or 1 µM). UT, untreated. (E) qRT-PCR determination of Atm .

    Journal: Cancer cell

    Article Title: Mutant IDH1 downregulates ATM and alters DNA repair and sensitivity to DNA damage independent of TET2

    doi: 10.1016/j.ccell.2016.05.018

    Figure Lengend Snippet: Partially closed chromatin structure and increased H3K methylation in the ATM promoter in Vav-IDH1-KI cells (A) ATAC-qPCR to analyze the indicated genes in LSK cells from Vav-IDH1-KI and WT mice (n=3). Data are the mean ± SD. (B) Immunoblot to detect the indicated methylated forms of histone H3 in Lin − cells from Vav-IDH1-KI and WT mice. (C) ChIP-qPCR analysis of the Atm promoter in LSK cells from Vav-IDH1-KI and WT mice (n=3) using anti-histone H3-K9me3 Ab. Data are the mean ± SD. (D) Immunoblot to detect the indicated proteins in LSK cells isolated from Vav-IDH1-KI and WT mice and cultured with/without UNC1999 (1 µM) or UNC0642(0.25 or 1 µM). UT, untreated. (E) qRT-PCR determination of Atm .

    Article Snippet: Primary antibodies (Abs) used in this study recognized: ATM (Genetex, Cat.No: GTX70103 or Santa Cruz, Cat.No: sc-23921), phospho-ATM (Rockland, Cat.No: 200–301–400), Chek2 (Millipore, Cat.No: 05–649), phospho-p53 (Ser18) (Cell Signaling Technology, Cat. No: 9284), p53 (Vector Laboratories, Cat. No: VP-P956), p21 (BD Pharmingen, Cat.No: 556431), β-actin (Sigma; Cat.No: A2066), histone H3 (Abcam, Cat.No: ab10799), histone H3 (trimethyl K9) (Abcam, Cat.No: ab8898), histone H3 (dimethyl K9) (Abcam, Cat.No: ab1220), histone H3 (trimethyl K27) (Millipore, Cat.No: 07–449), histone H3 (dimethyl K79) (Cell Signalling Technology, Cat.No: 9757), and histone H3 (trimethyl K36) (Abcam, Cat.No: ab9050).

    Techniques: Methylation, Real-time Polymerase Chain Reaction, Mouse Assay, Chromatin Immunoprecipitation, Isolation, Cell Culture, Quantitative RT-PCR

    Proximity biotin ligation assay of KIF2A isoforms in Neuro2A cells. A . Immunofluorescence staining of myc (red), Streptavidin (green) and DNA (blue) in Neuro2A cells transfected with myc BirA* , myc BirA*-Kif2a.1, mycBirA*-Kif2a.2, mycBirA*-Kif2a.3  or  mycBirA*-Kif2a H321D  separately. Transfected cells were identified based on myc immunostaining. Scale bar, 10 µm.  B . Top panel: Immunoblotting with anti-myc showing expression of mycBirA*-KIF2A in Neuro2A cells. Middle panel: Immunoblotting with anti-biotin confirming biotinylation. Bottom panel: Anti-Histone H3 as loading control.  C . Pull-down with streptavidin beads followed by anti-biotin immunoblotting of Neuro2A cell lysates. Biotin supplementation to cell media is indicated.

    Journal: bioRxiv

    Article Title: Roles of developmentally regulated KIF2A alternative isoforms in cortical neuron migration and differentiation

    doi: 10.1101/2020.05.07.081216

    Figure Lengend Snippet: Proximity biotin ligation assay of KIF2A isoforms in Neuro2A cells. A . Immunofluorescence staining of myc (red), Streptavidin (green) and DNA (blue) in Neuro2A cells transfected with myc BirA* , myc BirA*-Kif2a.1, mycBirA*-Kif2a.2, mycBirA*-Kif2a.3 or mycBirA*-Kif2a H321D separately. Transfected cells were identified based on myc immunostaining. Scale bar, 10 µm. B . Top panel: Immunoblotting with anti-myc showing expression of mycBirA*-KIF2A in Neuro2A cells. Middle panel: Immunoblotting with anti-biotin confirming biotinylation. Bottom panel: Anti-Histone H3 as loading control. C . Pull-down with streptavidin beads followed by anti-biotin immunoblotting of Neuro2A cell lysates. Biotin supplementation to cell media is indicated.

    Article Snippet: SC-40, 1:1000); hnRNP C1+C2 (Abcam, cat.no. ab10294, 1:2000); biotin (gift from Dr Timothy J Mitchison, Harvard Medical School, Boston, MA, 1:20,000); Histone H3 (Cell Signaling Technology, cat.no.

    Techniques: Ligation, Immunofluorescence, Staining, Transfection, Immunostaining, Expressing

    Pharmacological inhibition of IKK β inhibits adipocyte differentiation. (A) Oil red O staining of 3T3-L1 cells induced by differentiation medium or medium containing IKKβ inhibitor BMS-345541 at indicated concentrations. (B) Analysis of IKKβ, Smurf2, and adipogenic genes in 3T3-L1 cells treated with control or 10 µM BMS-345541 for 48 h by QPCR ( n = 3–5). (C) 3T3-L1 cells were treated with vehicle control or 10 µM BMS-345541 for 48 h before incubating with vehicle control or 100 nM PS-341 for 4 h. β-catenin was immunoprecipitated with anti–β-catenin antibodies, and then probed with antiubiquitin monoclonal antibodies. The whole-cell lysates were probed with anti–β-catenin antibodies as an internal control. (D) Western blot analysis of nuclear β-catenin levels in 3T3-L1 cells treated with vehicle control or 10 µM BMS-345541 for 48 h. Nuclear proteins were probed with anti-Histone H3 antibodies as an internal control. (E) Oil red O staining of 3T3-L1 cells induced by differentiation medium or medium containing IKKβ inhibitor sodium salicylate at indicated concentrations. Similar results were obtained from at least three independent experiments. All data are mean ± SD. **, P

    Journal: The Journal of Experimental Medicine

    Article Title: IKKβ links vascular inflammation to obesity and atherosclerosis

    doi: 10.1084/jem.20131281

    Figure Lengend Snippet: Pharmacological inhibition of IKK β inhibits adipocyte differentiation. (A) Oil red O staining of 3T3-L1 cells induced by differentiation medium or medium containing IKKβ inhibitor BMS-345541 at indicated concentrations. (B) Analysis of IKKβ, Smurf2, and adipogenic genes in 3T3-L1 cells treated with control or 10 µM BMS-345541 for 48 h by QPCR ( n = 3–5). (C) 3T3-L1 cells were treated with vehicle control or 10 µM BMS-345541 for 48 h before incubating with vehicle control or 100 nM PS-341 for 4 h. β-catenin was immunoprecipitated with anti–β-catenin antibodies, and then probed with antiubiquitin monoclonal antibodies. The whole-cell lysates were probed with anti–β-catenin antibodies as an internal control. (D) Western blot analysis of nuclear β-catenin levels in 3T3-L1 cells treated with vehicle control or 10 µM BMS-345541 for 48 h. Nuclear proteins were probed with anti-Histone H3 antibodies as an internal control. (E) Oil red O staining of 3T3-L1 cells induced by differentiation medium or medium containing IKKβ inhibitor sodium salicylate at indicated concentrations. Similar results were obtained from at least three independent experiments. All data are mean ± SD. **, P

    Article Snippet: Anti-IKKβ, anti-IKKα, anti-Smurf2, and anti-Histone H3 antibodies were purchased from Cell Signaling Technology; anti–β-catenin, anti–β-actin, and anti-GAPDH antibodies were purchased from Sigma-Aldrich; and antiubiquitin monoclonal antibody was purchased from Santa Cruz Biotechnology.

    Techniques: Inhibition, Staining, Real-time Polymerase Chain Reaction, Immunoprecipitation, Western Blot

    Chronic treatment of mice with IKKβ inhibitor ameliorates diet-induced obesity. (A and B) 8-wk-old male C57BL/6 mice were fed a WD and treated with vehicle or 10 mg/kg body weight of BMS-345541 by daily oral gavage for 8 wk. Body weight (A) was measured weekly and fat and lean mass (B) were measured at the end of feeding study ( n = 11–12 mice). (C) Representative photographs of subcutaneous (Sub) and epididymal (Epi) WAT. (D) Expression of Smurf2 and other NF-κB target genes in WAT were analyzed by QPCR ( n = 4 mice). (E) Western blot analysis of nuclear β-catenin levels in WAT of control or BMS-345541-treated mice. Nuclear proteins were probed with anti-Histone H3 antibodies an internal control. (F) Oil red O staining of adipose SV cells from control or BMS-345541–treated mice induced by differentiation medium. (G) Adipose SV cells isolated from control or BMS-345541-treated mice were incubated with vehicle or 100 nM PS-341 as indicated for 4 h. β-catenin was immunoprecipitated with anti–β-catenin antibodies and then probed with antiubiquitin monoclonal antibodies. The whole cell lysates were probed with anti–β-catenin antibodies as an internal control. Similar results were obtained from at least three independent experiments. All data are mean ± SD. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: IKKβ links vascular inflammation to obesity and atherosclerosis

    doi: 10.1084/jem.20131281

    Figure Lengend Snippet: Chronic treatment of mice with IKKβ inhibitor ameliorates diet-induced obesity. (A and B) 8-wk-old male C57BL/6 mice were fed a WD and treated with vehicle or 10 mg/kg body weight of BMS-345541 by daily oral gavage for 8 wk. Body weight (A) was measured weekly and fat and lean mass (B) were measured at the end of feeding study ( n = 11–12 mice). (C) Representative photographs of subcutaneous (Sub) and epididymal (Epi) WAT. (D) Expression of Smurf2 and other NF-κB target genes in WAT were analyzed by QPCR ( n = 4 mice). (E) Western blot analysis of nuclear β-catenin levels in WAT of control or BMS-345541-treated mice. Nuclear proteins were probed with anti-Histone H3 antibodies an internal control. (F) Oil red O staining of adipose SV cells from control or BMS-345541–treated mice induced by differentiation medium. (G) Adipose SV cells isolated from control or BMS-345541-treated mice were incubated with vehicle or 100 nM PS-341 as indicated for 4 h. β-catenin was immunoprecipitated with anti–β-catenin antibodies and then probed with antiubiquitin monoclonal antibodies. The whole cell lysates were probed with anti–β-catenin antibodies as an internal control. Similar results were obtained from at least three independent experiments. All data are mean ± SD. *, P

    Article Snippet: Anti-IKKβ, anti-IKKα, anti-Smurf2, and anti-Histone H3 antibodies were purchased from Cell Signaling Technology; anti–β-catenin, anti–β-actin, and anti-GAPDH antibodies were purchased from Sigma-Aldrich; and antiubiquitin monoclonal antibody was purchased from Santa Cruz Biotechnology.

    Techniques: Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining, Isolation, Incubation, Immunoprecipitation

    IKKβ regulates β-catenin ubiquitination and adipocyte differentiation. (A) Expression levels of preadipocyte, mural cell, and mature adipocyte markers in 3T3-L1 cells at 0 and 48 h after addition of differentiation media ( n = 3). (B) Western blot analysis of IKKβ and IKKα levels in 3T3-L1 preadipocytes expressing control shRNA or shRNA against IKKβ (shIKKβ). (C) Oil red O staining of control or shIKKβ 3T3-L1 cells induced by differentiation medium. (D) Western blot analysis of nuclear β-catenin protein levels of control or shIKKβ 3T3-L1 cells. Nuclear proteins were also probed with anti-Histone H3 antibodies as an internal control. (E) QPCR analysis of expression of IKKβ and adipogenic genes in control or shIKKβ 3T3-L1 cells ( n = 3–5). (F) Control or shIKKβ 3T3-L1 cells were treated with vehicle control or 100 nM PS-341 as indicated for 4 h. β-catenin was immunoprecipitated with anti–β-catenin antibodies, and then probed with antiubiquitin monoclonal antibodies. The whole cell lysates were probed with anti–β-catenin antibodies as an internal control. (G) QPCR analysis of Smurf2 expression in control or shIKKβ 3T3-L1 cells ( n = 4). (H) Western blot analysis of Smurf2 protein levels in control or shIKKβ 3T3-L1 cells. (I) Oil red O staining of 3T3-L1 cells transfected with control vector or vector expressing IκBαM induced by differentiation medium. (J) Western blot analysis of Smurf2 protein levels in 3T3-L1 cells expressing control or IκBαM vectors. (K) Control or IκBαM-expressing 3T3-L1 cells were treated with vehicle control or 100 nM PS-341 as indicated for 4 h. β-catenin was immunoprecipitated with anti–β-catenin antibodies and then probed with anti-ubiquitin monoclonal antibodies. The whole-cell lysates were probed with anti–β-catenin antibodies as an internal control. (L) Western blot analysis of nuclear β-catenin levels in control or IκBαM-expressing 3T3-L1 cells. Nuclear proteins were probed with anti-Histone H3 antibodies as an internal control. (M) Western blot analysis of Smurf2 levels in adipose SV cells isolated from WD-fed IKKβ F/F LDLR −/− (F/F) and SM22Cre + IKKβ F/F LDLR −/− (KO) mice. (N) Adipose SV cells were treated with vehicle control or 100 nM PS-341, as indicated, for 4 h. β-catenin was immunoprecipitated with anti–β-catenin antibodies and then probed with anti-ubiquitin monoclonal antibodies. The whole cell lysates were probed with anti-β-catenin antibodies as an internal control. (O) Western blot analysis of nuclear β-catenin levels in SV cells. Nuclear proteins were probed with anti-Histone H3 antibodies as internal control. Similar results were obtained from at least three independent experiments. All data are means ± SD. **, P

    Journal: The Journal of Experimental Medicine

    Article Title: IKKβ links vascular inflammation to obesity and atherosclerosis

    doi: 10.1084/jem.20131281

    Figure Lengend Snippet: IKKβ regulates β-catenin ubiquitination and adipocyte differentiation. (A) Expression levels of preadipocyte, mural cell, and mature adipocyte markers in 3T3-L1 cells at 0 and 48 h after addition of differentiation media ( n = 3). (B) Western blot analysis of IKKβ and IKKα levels in 3T3-L1 preadipocytes expressing control shRNA or shRNA against IKKβ (shIKKβ). (C) Oil red O staining of control or shIKKβ 3T3-L1 cells induced by differentiation medium. (D) Western blot analysis of nuclear β-catenin protein levels of control or shIKKβ 3T3-L1 cells. Nuclear proteins were also probed with anti-Histone H3 antibodies as an internal control. (E) QPCR analysis of expression of IKKβ and adipogenic genes in control or shIKKβ 3T3-L1 cells ( n = 3–5). (F) Control or shIKKβ 3T3-L1 cells were treated with vehicle control or 100 nM PS-341 as indicated for 4 h. β-catenin was immunoprecipitated with anti–β-catenin antibodies, and then probed with antiubiquitin monoclonal antibodies. The whole cell lysates were probed with anti–β-catenin antibodies as an internal control. (G) QPCR analysis of Smurf2 expression in control or shIKKβ 3T3-L1 cells ( n = 4). (H) Western blot analysis of Smurf2 protein levels in control or shIKKβ 3T3-L1 cells. (I) Oil red O staining of 3T3-L1 cells transfected with control vector or vector expressing IκBαM induced by differentiation medium. (J) Western blot analysis of Smurf2 protein levels in 3T3-L1 cells expressing control or IκBαM vectors. (K) Control or IκBαM-expressing 3T3-L1 cells were treated with vehicle control or 100 nM PS-341 as indicated for 4 h. β-catenin was immunoprecipitated with anti–β-catenin antibodies and then probed with anti-ubiquitin monoclonal antibodies. The whole-cell lysates were probed with anti–β-catenin antibodies as an internal control. (L) Western blot analysis of nuclear β-catenin levels in control or IκBαM-expressing 3T3-L1 cells. Nuclear proteins were probed with anti-Histone H3 antibodies as an internal control. (M) Western blot analysis of Smurf2 levels in adipose SV cells isolated from WD-fed IKKβ F/F LDLR −/− (F/F) and SM22Cre + IKKβ F/F LDLR −/− (KO) mice. (N) Adipose SV cells were treated with vehicle control or 100 nM PS-341, as indicated, for 4 h. β-catenin was immunoprecipitated with anti–β-catenin antibodies and then probed with anti-ubiquitin monoclonal antibodies. The whole cell lysates were probed with anti-β-catenin antibodies as an internal control. (O) Western blot analysis of nuclear β-catenin levels in SV cells. Nuclear proteins were probed with anti-Histone H3 antibodies as internal control. Similar results were obtained from at least three independent experiments. All data are means ± SD. **, P

    Article Snippet: Anti-IKKβ, anti-IKKα, anti-Smurf2, and anti-Histone H3 antibodies were purchased from Cell Signaling Technology; anti–β-catenin, anti–β-actin, and anti-GAPDH antibodies were purchased from Sigma-Aldrich; and antiubiquitin monoclonal antibody was purchased from Santa Cruz Biotechnology.

    Techniques: Expressing, Western Blot, shRNA, Staining, Real-time Polymerase Chain Reaction, Immunoprecipitation, Transfection, Plasmid Preparation, Isolation, Mouse Assay

    The effect of oncoVV-HddSBL on histone modification. C6 glioblastoma cells were treated with PBS, 5 MOI of oncoVV or oncoVV-HddSBL, and histone H3, H4, H3R8, and H4R3 asymmetric dimethylation levels, as well as the expression of FLAG-tagged HddSBL were analyzed by Western blot.

    Journal: Marine Drugs

    Article Title: Haliotis discus discus Sialic Acid-Binding Lectin Reduces the Oncolytic Vaccinia Virus Induced Toxicity in a Glioblastoma Mouse Model

    doi: 10.3390/md16050141

    Figure Lengend Snippet: The effect of oncoVV-HddSBL on histone modification. C6 glioblastoma cells were treated with PBS, 5 MOI of oncoVV or oncoVV-HddSBL, and histone H3, H4, H3R8, and H4R3 asymmetric dimethylation levels, as well as the expression of FLAG-tagged HddSBL were analyzed by Western blot.

    Article Snippet: Anti-histone H3, anti-histone H4, anti-flag, and anti-β-actin antibodies were obtained from Cell Signaling Technology Inc. (Danvers, MA, USA).

    Techniques: Modification, Expressing, Western Blot

    TGF-β-associated gene expression profiles induced by treatment with mitogen-activated protein kinase inhibitors. (A) CRL-2097 human dermal fibroblasts were cultured for 15-60 min in the presence or absence of 10 µ M U0126 (ERK i ) or 10 µ M SP600125 (JNK i ) and subjected to western blot analysis for p-ERK1/2. Histone H3 was used as a loading control. Fibroblasts incubated with recombinant human FGF2 for 30 min were used as a positive control for p-ERK1/2 expression. (B) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ) or 10 µ M SB202190 (p38 i ) until day 4. Expression levels of TGF-β-associated transcripts TGFB1 , TGFB2 , TGFBR1 , TGFBR2 and TGFBR3 were determined relative to fibroblasts cultured under control conditions by reverse transcription-quantitative polymerase chain reaction. GAPDH expression was used as an internal control. Expression levels per transcript were compared using an one-way analysis of variance and post-hoc Holm-Sidak analysis. * P

    Journal: International Journal of Molecular Medicine

    Article Title: Crosstalk between mitogen-activated protein kinase inhibitors and transforming growth factor-β signaling results in variable activation of human dermal fibroblasts

    doi: 10.3892/ijmm.2018.3949

    Figure Lengend Snippet: TGF-β-associated gene expression profiles induced by treatment with mitogen-activated protein kinase inhibitors. (A) CRL-2097 human dermal fibroblasts were cultured for 15-60 min in the presence or absence of 10 µ M U0126 (ERK i ) or 10 µ M SP600125 (JNK i ) and subjected to western blot analysis for p-ERK1/2. Histone H3 was used as a loading control. Fibroblasts incubated with recombinant human FGF2 for 30 min were used as a positive control for p-ERK1/2 expression. (B) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ) or 10 µ M SB202190 (p38 i ) until day 4. Expression levels of TGF-β-associated transcripts TGFB1 , TGFB2 , TGFBR1 , TGFBR2 and TGFBR3 were determined relative to fibroblasts cultured under control conditions by reverse transcription-quantitative polymerase chain reaction. GAPDH expression was used as an internal control. Expression levels per transcript were compared using an one-way analysis of variance and post-hoc Holm-Sidak analysis. * P

    Article Snippet: Reagents The primary antibodies used were the following: α-SMA (cat. no. sc-32251; 1:1,000 for western blotting, 1:100 for fluorescence microscopy), collagen I (cat. no. sc-8783; 1:100), ED-A fibronectin (ED-A Fn; cat. no. sc-59826; 1:100), Histone H3 (cat. no. sc-8654-R; 1:1,000) (all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA), Calponin (clone: CALP; 1:1,000; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA), SM22α (cat. no. VPA00048KT; 1:1,000; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and p-ERK1/2 (cat. no. 4370; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA).

    Techniques: Expressing, Cell Culture, Western Blot, Incubation, Recombinant, Positive Control, Real-time Polymerase Chain Reaction

    Effects of TGF-βR1 inhibition on fibroblast activation induced by ERK or JNK inhibitors. (A) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of the indicated concentration of the TGF-βR1 inhibitor RepSox until day 4 and then subjected to Western blot analysis for myofibroblast markers α-SMA and calponin. Histone H3 was used as a loading control. (B) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ), 10 ng/ml TGF-β1 and 100 nM RepSox until day 4 and then subjected to western blot analysis for myofibroblast marker α-SMA. Histone H3 was used as a loading control. (C) Western blots were analyzed using densitometry, and relative density of α-SMA/Histone H3 was compared for each mitogen-activated protein kinase inhibitor-treated or TGF-β1-treated sample compared with its corresponding TGF-βR1 inhibitor-treated sample. Statistical significance was determined using a two-tailed Student’s t-test. * P

    Journal: International Journal of Molecular Medicine

    Article Title: Crosstalk between mitogen-activated protein kinase inhibitors and transforming growth factor-β signaling results in variable activation of human dermal fibroblasts

    doi: 10.3892/ijmm.2018.3949

    Figure Lengend Snippet: Effects of TGF-βR1 inhibition on fibroblast activation induced by ERK or JNK inhibitors. (A) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of the indicated concentration of the TGF-βR1 inhibitor RepSox until day 4 and then subjected to Western blot analysis for myofibroblast markers α-SMA and calponin. Histone H3 was used as a loading control. (B) CRL-2097 human dermal fibroblasts were cultured in the presence or absence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ), 10 ng/ml TGF-β1 and 100 nM RepSox until day 4 and then subjected to western blot analysis for myofibroblast marker α-SMA. Histone H3 was used as a loading control. (C) Western blots were analyzed using densitometry, and relative density of α-SMA/Histone H3 was compared for each mitogen-activated protein kinase inhibitor-treated or TGF-β1-treated sample compared with its corresponding TGF-βR1 inhibitor-treated sample. Statistical significance was determined using a two-tailed Student’s t-test. * P

    Article Snippet: Reagents The primary antibodies used were the following: α-SMA (cat. no. sc-32251; 1:1,000 for western blotting, 1:100 for fluorescence microscopy), collagen I (cat. no. sc-8783; 1:100), ED-A fibronectin (ED-A Fn; cat. no. sc-59826; 1:100), Histone H3 (cat. no. sc-8654-R; 1:1,000) (all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA), Calponin (clone: CALP; 1:1,000; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA), SM22α (cat. no. VPA00048KT; 1:1,000; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and p-ERK1/2 (cat. no. 4370; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA).

    Techniques: Inhibition, Activation Assay, Cell Culture, Concentration Assay, Western Blot, Marker, Two Tailed Test

    Cooperative effects of mitogen-activated protein kinase inhibition and exogenous TGF-β on fibroblast activation. CRL-2097 human dermal fibroblasts were cultured under control conditions or in the presence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ), and/or 10 ng/ml exogenous recombinant human TGF-β1, and analyzed at day 4 in culture. (A) Protein lysates were examined for expression of myofibroblast-associated protein markers α-SMA, calponin, and SM22α by western blot analysis. Histone H3 was used as a loading control. (B) Fibroblasts were fixed and stained for the myofibroblast marker protein α-SMA and nuclei were counterstained with Hoechst. Scale bar, 100 µ m. TGF-β, transforming growth factor-β; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; α-SMA, smooth muscle α actin; i , inhibitor.

    Journal: International Journal of Molecular Medicine

    Article Title: Crosstalk between mitogen-activated protein kinase inhibitors and transforming growth factor-β signaling results in variable activation of human dermal fibroblasts

    doi: 10.3892/ijmm.2018.3949

    Figure Lengend Snippet: Cooperative effects of mitogen-activated protein kinase inhibition and exogenous TGF-β on fibroblast activation. CRL-2097 human dermal fibroblasts were cultured under control conditions or in the presence of 10 µ M U0126 (ERK i ), 10 µ M SP600125 (JNK i ), and/or 10 ng/ml exogenous recombinant human TGF-β1, and analyzed at day 4 in culture. (A) Protein lysates were examined for expression of myofibroblast-associated protein markers α-SMA, calponin, and SM22α by western blot analysis. Histone H3 was used as a loading control. (B) Fibroblasts were fixed and stained for the myofibroblast marker protein α-SMA and nuclei were counterstained with Hoechst. Scale bar, 100 µ m. TGF-β, transforming growth factor-β; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; α-SMA, smooth muscle α actin; i , inhibitor.

    Article Snippet: Reagents The primary antibodies used were the following: α-SMA (cat. no. sc-32251; 1:1,000 for western blotting, 1:100 for fluorescence microscopy), collagen I (cat. no. sc-8783; 1:100), ED-A fibronectin (ED-A Fn; cat. no. sc-59826; 1:100), Histone H3 (cat. no. sc-8654-R; 1:1,000) (all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA), Calponin (clone: CALP; 1:1,000; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA), SM22α (cat. no. VPA00048KT; 1:1,000; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and p-ERK1/2 (cat. no. 4370; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA).

    Techniques: Inhibition, Activation Assay, Cell Culture, Recombinant, Expressing, Western Blot, Staining, Marker

    Histone acetylation and E2F1 binding at the BRCA1 and RAD51 promoter regions. ( A, B ) CBP and p300 recruitment to the BRCA1 and RAD51 promoter regions. H1299 cells were subjected to ChIP using either control IgG or antibodies against CBP and p300. Immunoprecipitated DNAs were subjected to quantitative PCR for the detection of the promoter region of BRCA1 (A) or RAD51 (B) and for the detection of the non-promoter region of GAPDH . Normalized ChIP enrichment was calculated to show the degree of enrichment following pulldown with anti-p300 and anti-CBP antibodies. First, ChIP enrichment at the test sites ( BRCA1 or RAD51 promoter and the GAPDH gene body) was calculated as the fraction of total input DNA that was pulled down by the specific antibody or the non-specific control IgG. Then, normalized ChIP enrichment at the test sites was calculated by dividing the ChIP enrichment of the specific antibody by that of the non-specific control IgG. Data represent mean values ± SD. ( C–J ) Impaired histone acetylation and changes in E2F1 and E2F4 binding at BRCA1 and RAD51 promoter regions caused by knockdown of CBP and p300. H1299 cells pre-transfected with non-targeting (siNT) or CBP and p300 (siC+p) siRNAs were subjected to ChIP assays. DNA immunoprecipitated with anti-acetylated H3 K18 (H3 AceK18) (C, D), anti-acetylated H4 (H4 AceK5/8/12/16) (E, F), anti-E2F1 (G, H), or anti-E2F4 (I, J) antibodies was subjected to quantitative PCR to detect the promoter regions of BRCA1 (C, E, G, I) and RAD51 (D, F, H, J), and the gene body region of GAPDH . Relative ChIP enrichment was calculated to show the decrease/increase in enrichment at these sites following gene knockdown. The relative ChIP enrichment was calculated by dividing the ChIP enrichment at the BRCA1 or RAD51 promoter and the GAPDH gene body region in the siRNA-treated sample by the ChIP enrichment at the BRCA1 or RAD51 promoter in the siNT-treated sample. Data represent the mean ± SD.

    Journal: PLoS ONE

    Article Title: CBP and p300 Histone Acetyltransferases Contribute to Homologous Recombination by Transcriptionally Activating the BRCA1 and RAD51 Genes

    doi: 10.1371/journal.pone.0052810

    Figure Lengend Snippet: Histone acetylation and E2F1 binding at the BRCA1 and RAD51 promoter regions. ( A, B ) CBP and p300 recruitment to the BRCA1 and RAD51 promoter regions. H1299 cells were subjected to ChIP using either control IgG or antibodies against CBP and p300. Immunoprecipitated DNAs were subjected to quantitative PCR for the detection of the promoter region of BRCA1 (A) or RAD51 (B) and for the detection of the non-promoter region of GAPDH . Normalized ChIP enrichment was calculated to show the degree of enrichment following pulldown with anti-p300 and anti-CBP antibodies. First, ChIP enrichment at the test sites ( BRCA1 or RAD51 promoter and the GAPDH gene body) was calculated as the fraction of total input DNA that was pulled down by the specific antibody or the non-specific control IgG. Then, normalized ChIP enrichment at the test sites was calculated by dividing the ChIP enrichment of the specific antibody by that of the non-specific control IgG. Data represent mean values ± SD. ( C–J ) Impaired histone acetylation and changes in E2F1 and E2F4 binding at BRCA1 and RAD51 promoter regions caused by knockdown of CBP and p300. H1299 cells pre-transfected with non-targeting (siNT) or CBP and p300 (siC+p) siRNAs were subjected to ChIP assays. DNA immunoprecipitated with anti-acetylated H3 K18 (H3 AceK18) (C, D), anti-acetylated H4 (H4 AceK5/8/12/16) (E, F), anti-E2F1 (G, H), or anti-E2F4 (I, J) antibodies was subjected to quantitative PCR to detect the promoter regions of BRCA1 (C, E, G, I) and RAD51 (D, F, H, J), and the gene body region of GAPDH . Relative ChIP enrichment was calculated to show the decrease/increase in enrichment at these sites following gene knockdown. The relative ChIP enrichment was calculated by dividing the ChIP enrichment at the BRCA1 or RAD51 promoter and the GAPDH gene body region in the siRNA-treated sample by the ChIP enrichment at the BRCA1 or RAD51 promoter in the siNT-treated sample. Data represent the mean ± SD.

    Article Snippet: These results indicated that CBP and p300 directly bind to the BRCA1 and RAD51 promoter regions. p300 acetylates lysines 14 and 18 of histone H3, and lysines 5 and 8 of histone H4 in vitro , , .

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Transfection

    Impairment of RPA foci formation and the DNA damage checkpoint in CBP and p300 depleted cells. ( A ) Effects of CBP and p300 depletion on DSB-induced RPA and CHK1 activation. H1299 cells were pre-transfected with non-targeting (siNT) or CBP and p300 (siC+p) siRNA for 48 hr, and treated with DMSO (NT), 1 μM camptothecin (CPT), or 500 ng/ml neocarzinostatin (NCS) for 2 hr. Whole cell extracts were subjected to immunoblot analysis. ( B, C ) Decreased DSB-induced chromatin binding of RPA proteins. Chromatin enriched fractions from H1299 cells pre-transfected for 48 hr with non-targeting (siNT), BRCA1 (B), or CBP and p300 (siC+p) (C) siRNAs and treated with DMSO (NT) or 1 μM camptothecin (CPT) for 2 hr were subjected to immunoblot analysis. ( D, E ) Decreased DNA damage-induced chromatin binding of RPA proteins in H1299 cells. Chromatin enriched fractions from cells pre-transfected for 48 hr with non-targeting (siNT) or CBP+p300 (siC+p) siRNA were treated or not treated (NT) with (D) 500 ng/ml neocarzinostatin (NCS) for 1 or 2 hr, or (E) incubated for 3 hr after 50 Gy ionizing irradiation (IR) and subjected to immunoblot analysis. ( F, G ) Decreased DSB-induced RPA32 foci formation. H1299 cells were transfected with non-targeting (siNT) or CBP and p300 (siC+p) siRNA for 48 hr, treated with DMSO (NT) or 1 μM camptothecin (CPT) for 3 hr, and subjected to immunofluorescence analysis. The percentages of cells with RPA32 foci (F) or phosphorylated RPA32 (pS4/S8) foci (G) are shown. Data represent the mean ± SD. ( H, I ) CPT- and IR-induced formation of BRCA1 foci. H1299 cells were transfected with non-targeting (siNT) or CBP and p300 (siC+p) siRNA for 48 hr, treated with 1 μM camptothecin (CPT) for 3 hr (H) or 10 Gy ionizing irradiation (IR) for 4 hr (I), and subjected to immunofluorescence analysis. The percentages of cells showing  > 10 BRCA1 foci are shown. Data represent the mean ± SD. ( J ) Suppression of the G2/M DNA damage checkpoint. H1299 cells were pre-transfected for with non-targeting (siNT) or CBP and p300 (siC+p) siRNA for 48 hr. The cells were then irradiated (IR, 3 Gy) for 1 hr or not irradiated (No IR), and stained with anti-phospho-histone H3 antibody and propidium iodide (PI). The percentage of mitotic cells (mitotic index) was determined by flow cytometry. Data represent the mean ± SD. ( K ) Colony formation of H1299 cells treated with CPT. H1299 cells were pre-transfected for with non-targeting (siNT) or CBP and p300 (siC+p) siRNA for 48 hr. siRNA-treated cells were replated and incubated for 10 days. Survival is expressed as a percentage representing the number of colonies in treated samples relative to the number in DMSO-treated samples.

    Journal: PLoS ONE

    Article Title: CBP and p300 Histone Acetyltransferases Contribute to Homologous Recombination by Transcriptionally Activating the BRCA1 and RAD51 Genes

    doi: 10.1371/journal.pone.0052810

    Figure Lengend Snippet: Impairment of RPA foci formation and the DNA damage checkpoint in CBP and p300 depleted cells. ( A ) Effects of CBP and p300 depletion on DSB-induced RPA and CHK1 activation. H1299 cells were pre-transfected with non-targeting (siNT) or CBP and p300 (siC+p) siRNA for 48 hr, and treated with DMSO (NT), 1 μM camptothecin (CPT), or 500 ng/ml neocarzinostatin (NCS) for 2 hr. Whole cell extracts were subjected to immunoblot analysis. ( B, C ) Decreased DSB-induced chromatin binding of RPA proteins. Chromatin enriched fractions from H1299 cells pre-transfected for 48 hr with non-targeting (siNT), BRCA1 (B), or CBP and p300 (siC+p) (C) siRNAs and treated with DMSO (NT) or 1 μM camptothecin (CPT) for 2 hr were subjected to immunoblot analysis. ( D, E ) Decreased DNA damage-induced chromatin binding of RPA proteins in H1299 cells. Chromatin enriched fractions from cells pre-transfected for 48 hr with non-targeting (siNT) or CBP+p300 (siC+p) siRNA were treated or not treated (NT) with (D) 500 ng/ml neocarzinostatin (NCS) for 1 or 2 hr, or (E) incubated for 3 hr after 50 Gy ionizing irradiation (IR) and subjected to immunoblot analysis. ( F, G ) Decreased DSB-induced RPA32 foci formation. H1299 cells were transfected with non-targeting (siNT) or CBP and p300 (siC+p) siRNA for 48 hr, treated with DMSO (NT) or 1 μM camptothecin (CPT) for 3 hr, and subjected to immunofluorescence analysis. The percentages of cells with RPA32 foci (F) or phosphorylated RPA32 (pS4/S8) foci (G) are shown. Data represent the mean ± SD. ( H, I ) CPT- and IR-induced formation of BRCA1 foci. H1299 cells were transfected with non-targeting (siNT) or CBP and p300 (siC+p) siRNA for 48 hr, treated with 1 μM camptothecin (CPT) for 3 hr (H) or 10 Gy ionizing irradiation (IR) for 4 hr (I), and subjected to immunofluorescence analysis. The percentages of cells showing > 10 BRCA1 foci are shown. Data represent the mean ± SD. ( J ) Suppression of the G2/M DNA damage checkpoint. H1299 cells were pre-transfected for with non-targeting (siNT) or CBP and p300 (siC+p) siRNA for 48 hr. The cells were then irradiated (IR, 3 Gy) for 1 hr or not irradiated (No IR), and stained with anti-phospho-histone H3 antibody and propidium iodide (PI). The percentage of mitotic cells (mitotic index) was determined by flow cytometry. Data represent the mean ± SD. ( K ) Colony formation of H1299 cells treated with CPT. H1299 cells were pre-transfected for with non-targeting (siNT) or CBP and p300 (siC+p) siRNA for 48 hr. siRNA-treated cells were replated and incubated for 10 days. Survival is expressed as a percentage representing the number of colonies in treated samples relative to the number in DMSO-treated samples.

    Article Snippet: These results indicated that CBP and p300 directly bind to the BRCA1 and RAD51 promoter regions. p300 acetylates lysines 14 and 18 of histone H3, and lysines 5 and 8 of histone H4 in vitro , , .

    Techniques: Recombinase Polymerase Amplification, Activation Assay, Transfection, Cycling Probe Technology, Binding Assay, Incubation, Irradiation, Immunofluorescence, Staining, Flow Cytometry, Cytometry

    Recovery of histone acetylation by overexpression of wt p300 HAT. HS27/vIRF cells were transfected with wt p300 (lane 3) or p300 ΔHAT mutant (lane 4). HS27/cDNA3 (lane 1) and HS27/vIRF (lane 2) were included as controls. Identical amounts of proteins from cell lysates were used for immunoblotting analysis with an antibody specific for the acetylated histone H4. The bottom panel shows the amount of cellular histone H4 protein in each lane, detected by an anti-H4 antibody. Arrows indicate the acetylated form of histone H4 (Ac-H4) or total histone H4 (H4). Numbers at left are molecular masses in kilodaltons.

    Journal: Molecular and Cellular Biology

    Article Title: Inhibition of p300 Histone Acetyltransferase by Viral Interferon Regulatory Factor

    doi:

    Figure Lengend Snippet: Recovery of histone acetylation by overexpression of wt p300 HAT. HS27/vIRF cells were transfected with wt p300 (lane 3) or p300 ΔHAT mutant (lane 4). HS27/cDNA3 (lane 1) and HS27/vIRF (lane 2) were included as controls. Identical amounts of proteins from cell lysates were used for immunoblotting analysis with an antibody specific for the acetylated histone H4. The bottom panel shows the amount of cellular histone H4 protein in each lane, detected by an anti-H4 antibody. Arrows indicate the acetylated form of histone H4 (Ac-H4) or total histone H4 (H4). Numbers at left are molecular masses in kilodaltons.

    Article Snippet: Purified full-length p300 was mixed with histone H4 and 3 H-labeled acetyl-CoA in the presence or absence of purified vIRF protein.

    Techniques: Over Expression, HAT Assay, Transfection, Mutagenesis

    Immunofluorescence test of in vivo histone H3 and H4 acetylation. HS27/cDNA3 and HS27/vIRF cells were stained with antibodies which specifically reacted with the acetylated forms of histones H3 and H4. Cells were visualized with Nomarski optics. Immunofluorescence testing was performed with a Leica confocal immunofluorescence microscope.

    Journal: Molecular and Cellular Biology

    Article Title: Inhibition of p300 Histone Acetyltransferase by Viral Interferon Regulatory Factor

    doi:

    Figure Lengend Snippet: Immunofluorescence test of in vivo histone H3 and H4 acetylation. HS27/cDNA3 and HS27/vIRF cells were stained with antibodies which specifically reacted with the acetylated forms of histones H3 and H4. Cells were visualized with Nomarski optics. Immunofluorescence testing was performed with a Leica confocal immunofluorescence microscope.

    Article Snippet: Purified full-length p300 was mixed with histone H4 and 3 H-labeled acetyl-CoA in the presence or absence of purified vIRF protein.

    Techniques: Immunofluorescence, In Vivo, Staining, Microscopy

    Inhibition of p300 HAT activity by vIRF. Recombinant baculovirus containing the flag-tagged p300, vIRF, or v-cyclin was used to purify each protein from insect cells. Purified p300 protein (30 nM) was mixed with [ 3 H]acetyl-CoA and histone H4 serving as substrates in the presence of increasing nanomolar amounts of vIRF or v-cyclin as indicated at the bottom of panel A. After 5 min, p300 HAT activity was measured by immunoblotting with an antibody specific for acetylated histone H4 (A) and quantitating radioactivity of 3 H-labeled histone H4 (B). In lane 7, p300 protein was first mixed with the substrates for 5 min, followed by incubation with vIRF protein (150 nM) for an additional 25 min. The bottom panel of panel A shows the amount of histone H4 protein used in each reaction, detected by an anti-H4 antibody. The values in panel B represent the averages of three independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Inhibition of p300 Histone Acetyltransferase by Viral Interferon Regulatory Factor

    doi:

    Figure Lengend Snippet: Inhibition of p300 HAT activity by vIRF. Recombinant baculovirus containing the flag-tagged p300, vIRF, or v-cyclin was used to purify each protein from insect cells. Purified p300 protein (30 nM) was mixed with [ 3 H]acetyl-CoA and histone H4 serving as substrates in the presence of increasing nanomolar amounts of vIRF or v-cyclin as indicated at the bottom of panel A. After 5 min, p300 HAT activity was measured by immunoblotting with an antibody specific for acetylated histone H4 (A) and quantitating radioactivity of 3 H-labeled histone H4 (B). In lane 7, p300 protein was first mixed with the substrates for 5 min, followed by incubation with vIRF protein (150 nM) for an additional 25 min. The bottom panel of panel A shows the amount of histone H4 protein used in each reaction, detected by an anti-H4 antibody. The values in panel B represent the averages of three independent experiments.

    Article Snippet: Purified full-length p300 was mixed with histone H4 and 3 H-labeled acetyl-CoA in the presence or absence of purified vIRF protein.

    Techniques: Inhibition, HAT Assay, Activity Assay, Recombinant, Purification, Radioactivity, Labeling, Incubation

    Alteration of in vivo histone H3 and H4 acetylation by vIRF expression or butyric acid treatment. Identical amounts of proteins from HS27/cDNA3 cells (lanes 1 and 3) and HS27/vIRF cells (lanes 2 and 4) treated with butyric acid overnight (lanes 3 and 4) or mock treated (lanes 1 and 2) were used for immunoblotting analysis with antibodies specific for the acetylated histone H3 (A) or H4 (B). Arrows indicate acetylated histones H3 (Ac-H3) and H4 (Ac-H4). Numbers at left of each panel show sizes in kilodaltons.

    Journal: Molecular and Cellular Biology

    Article Title: Inhibition of p300 Histone Acetyltransferase by Viral Interferon Regulatory Factor

    doi:

    Figure Lengend Snippet: Alteration of in vivo histone H3 and H4 acetylation by vIRF expression or butyric acid treatment. Identical amounts of proteins from HS27/cDNA3 cells (lanes 1 and 3) and HS27/vIRF cells (lanes 2 and 4) treated with butyric acid overnight (lanes 3 and 4) or mock treated (lanes 1 and 2) were used for immunoblotting analysis with antibodies specific for the acetylated histone H3 (A) or H4 (B). Arrows indicate acetylated histones H3 (Ac-H3) and H4 (Ac-H4). Numbers at left of each panel show sizes in kilodaltons.

    Article Snippet: Purified full-length p300 was mixed with histone H4 and 3 H-labeled acetyl-CoA in the presence or absence of purified vIRF protein.

    Techniques: In Vivo, Expressing

    GO/CDDP triggered direct association of histones H1/H4 and CDDP with LC3. (A) SDS-PAGE analysis of LC3-immunocomplex from the lysate (Lys) and nuclear (Nuc) fractions of cells with or without GO/CDDP treatment. (B) Western blot analysis of proteins (~11 kDa and ~35 kDa) derived from the nuclear LC3-immunocomplex. (C) Immunofluorescence microscopy of cells with or without GO/CDDP treatment. The cells were immunolabeled with primary antibody for histone H1 or H4 and counter stained with DAPI. (D) Percentages of cells positive with total histone H1, phosphorylated histone H1 (p-histone H1), histone H4 and acetylated histone H4 (H4K16ac). (E) Amount of CDDP associated with nuclear LC3 immunocomplex. The nuclear LC3 immunocomplex was analyzed by ICP-MS to quantify the amount of platinum (Pt) in CDDP. The data represent mean ± s.d of at least 3 independent culture experiments.

    Journal: Theranostics

    Article Title: Graphene oxide sensitizes cancer cells to chemotherapeutics by inducing early autophagy events, promoting nuclear trafficking and necrosis

    doi: 10.7150/thno.24173

    Figure Lengend Snippet: GO/CDDP triggered direct association of histones H1/H4 and CDDP with LC3. (A) SDS-PAGE analysis of LC3-immunocomplex from the lysate (Lys) and nuclear (Nuc) fractions of cells with or without GO/CDDP treatment. (B) Western blot analysis of proteins (~11 kDa and ~35 kDa) derived from the nuclear LC3-immunocomplex. (C) Immunofluorescence microscopy of cells with or without GO/CDDP treatment. The cells were immunolabeled with primary antibody for histone H1 or H4 and counter stained with DAPI. (D) Percentages of cells positive with total histone H1, phosphorylated histone H1 (p-histone H1), histone H4 and acetylated histone H4 (H4K16ac). (E) Amount of CDDP associated with nuclear LC3 immunocomplex. The nuclear LC3 immunocomplex was analyzed by ICP-MS to quantify the amount of platinum (Pt) in CDDP. The data represent mean ± s.d of at least 3 independent culture experiments.

    Article Snippet: The primary antibody was specific for LC3 (LC3B isoform, Cell Signaling, 3868), p62 (Abnova, H00008878), Lamp-2 (GeneTex, GTX13524), karyopherin alpha 2 (importin α) (BD Biosciences, cat no. 610485), karyopherin beta 2 (importin β) (BD Biosciences, cat no. 610559), acetyl-α-tubulin (Lys40) (Cell Signaling, cat no. 5335), histone H1 (Merck, cat no. 05-457), phosphorylated histone H1 (Merck, cat no. 06-597), acetylated histone H4 (H4K16ac, Genetex GTX632097) or histone H4 (D2X4V) (Cell Signaling, 13919).

    Techniques: SDS Page, Western Blot, Derivative Assay, Immunofluorescence, Microscopy, Immunolabeling, Staining, Mass Spectrometry

    Primer extension activity of RT HIV-1 using the 17mer/44mer primer/template duplex. The experiments were conducted for the times indicated in the figure (5–90 min) using undamaged templates (lanes 2–5), the template containing single, site-specific 1,2-GG intrastrand CL of cisplatin (lanes 6–9) and the template containing single DPCL formed by the transformation of the template containing site-specific 1,2-GG intrastrand CL of cisplatin incubated with histone H1 (lanes 10–13). Lane 1, 17-mer primer. The pause sites opposite the platinated guanines and the nucleotide preceding the platinated guanines (thymine residue on the 3′ side of the CL) are marked 34, 33, 32, respectively. The nucleotide sequences of the templates and the primers are shown beneath the gels. See the text for other details.

    Journal: Nucleic Acids Research

    Article Title: Mechanism of the formation of DNA-protein cross-links by antitumor cisplatin

    doi: 10.1093/nar/gkm032

    Figure Lengend Snippet: Primer extension activity of RT HIV-1 using the 17mer/44mer primer/template duplex. The experiments were conducted for the times indicated in the figure (5–90 min) using undamaged templates (lanes 2–5), the template containing single, site-specific 1,2-GG intrastrand CL of cisplatin (lanes 6–9) and the template containing single DPCL formed by the transformation of the template containing site-specific 1,2-GG intrastrand CL of cisplatin incubated with histone H1 (lanes 10–13). Lane 1, 17-mer primer. The pause sites opposite the platinated guanines and the nucleotide preceding the platinated guanines (thymine residue on the 3′ side of the CL) are marked 34, 33, 32, respectively. The nucleotide sequences of the templates and the primers are shown beneath the gels. See the text for other details.

    Article Snippet: No removal of the cisplatin adduct cross-linked to KF− or histone H1 from the 148 bp substrate by human or rodent excinuclease was observed under conditions when the 1,2-GG or 1,3 GTG intrastrand adducts (not cross-linked to a protein) were readily excised (shown in , lanes 4 and 5 for the substrate cross-linked to KF− ).

    Techniques: Activity Assay, Transformation Assay, Incubation

    Formation of DPCLs of unmodified and platinated 213-bp DNA fragment globally modified by cisplatin or transplatin ( r b = 0.025) with KF − and histone H1 assessed by agarose gel electrophoresis; the fragment was incubated with the protein for 24 h. Lanes: 1, 2, the fragment modified by cisplatin incubated with KF − , histone H1, respectively; 3, 4, the fragment modified by cisplatin incubated in the buffer used for reaction with KF − or histone H1, respectively, no protein added; 5, 6, the fragment modified by transplatin incubated with KF − , histone H1, respectively; 7, 8, the fragment modified by transplatin incubated in the buffer used for reaction with KF − or histone H1, respectively, no protein added; 9, 10, the unplatinated fragment incubated with KF − or histone H1, respectively. See the text for other details.

    Journal: Nucleic Acids Research

    Article Title: Mechanism of the formation of DNA-protein cross-links by antitumor cisplatin

    doi: 10.1093/nar/gkm032

    Figure Lengend Snippet: Formation of DPCLs of unmodified and platinated 213-bp DNA fragment globally modified by cisplatin or transplatin ( r b = 0.025) with KF − and histone H1 assessed by agarose gel electrophoresis; the fragment was incubated with the protein for 24 h. Lanes: 1, 2, the fragment modified by cisplatin incubated with KF − , histone H1, respectively; 3, 4, the fragment modified by cisplatin incubated in the buffer used for reaction with KF − or histone H1, respectively, no protein added; 5, 6, the fragment modified by transplatin incubated with KF − , histone H1, respectively; 7, 8, the fragment modified by transplatin incubated in the buffer used for reaction with KF − or histone H1, respectively, no protein added; 9, 10, the unplatinated fragment incubated with KF − or histone H1, respectively. See the text for other details.

    Article Snippet: No removal of the cisplatin adduct cross-linked to KF− or histone H1 from the 148 bp substrate by human or rodent excinuclease was observed under conditions when the 1,2-GG or 1,3 GTG intrastrand adducts (not cross-linked to a protein) were readily excised (shown in , lanes 4 and 5 for the substrate cross-linked to KF− ).

    Techniques: Modification, Agarose Gel Electrophoresis, Incubation

    Formation of DPCLs of unmodified and platinated oligodeoxyribonucleotide duplexes 40 bp (A and B) or NF-κB ( 20 ) (C) (see Figure 1 B for their nucleotide sequence) globally modified by cisplatin or transplatin ( r b = 0.025) with KF − (A), histone H1 (B) and NF-κB (C) assessed by SDS/PAA gel electrophoresis. Lanes: 1–3, the duplex modified by cisplatin incubated with the protein for 1, 4 and 24 h, respectively; 4–6, the duplex modified by transplatin incubated with the protein for 1, 4 and 24 h, respectively; 7, control, unplatinated duplex, no protein added; 8, control, unplatinated duplex incubated with the protein for 24 h; 9, control, duplex modified by cisplatin ( r b = 0.025), no protein added; 10, control, duplex modified by cisplatin ( r b = 0.025) incubated with the protein for 24 h. See the text for other details.

    Journal: Nucleic Acids Research

    Article Title: Mechanism of the formation of DNA-protein cross-links by antitumor cisplatin

    doi: 10.1093/nar/gkm032

    Figure Lengend Snippet: Formation of DPCLs of unmodified and platinated oligodeoxyribonucleotide duplexes 40 bp (A and B) or NF-κB ( 20 ) (C) (see Figure 1 B for their nucleotide sequence) globally modified by cisplatin or transplatin ( r b = 0.025) with KF − (A), histone H1 (B) and NF-κB (C) assessed by SDS/PAA gel electrophoresis. Lanes: 1–3, the duplex modified by cisplatin incubated with the protein for 1, 4 and 24 h, respectively; 4–6, the duplex modified by transplatin incubated with the protein for 1, 4 and 24 h, respectively; 7, control, unplatinated duplex, no protein added; 8, control, unplatinated duplex incubated with the protein for 24 h; 9, control, duplex modified by cisplatin ( r b = 0.025), no protein added; 10, control, duplex modified by cisplatin ( r b = 0.025) incubated with the protein for 24 h. See the text for other details.

    Article Snippet: No removal of the cisplatin adduct cross-linked to KF− or histone H1 from the 148 bp substrate by human or rodent excinuclease was observed under conditions when the 1,2-GG or 1,3 GTG intrastrand adducts (not cross-linked to a protein) were readily excised (shown in , lanes 4 and 5 for the substrate cross-linked to KF− ).

    Techniques: Sequencing, Modification, Nucleic Acid Electrophoresis, Incubation

    Cytotoxic effects of extracellular histones on alveolar epithelial cells. A ) LDH release from LA-4 and MLE-12 lung alveolar epithelial cells exposed to purified histones (50 μg/ml, 1 h), chromogenic assay. B ) [Ca 2+ ] i staining in untreated (Ctrl)

    Journal: The FASEB Journal

    Article Title: Extracellular histones are essential effectors of C5aR- and C5L2-mediated tissue damage and inflammation in acute lung injury

    doi: 10.1096/fj.13-236380

    Figure Lengend Snippet: Cytotoxic effects of extracellular histones on alveolar epithelial cells. A ) LDH release from LA-4 and MLE-12 lung alveolar epithelial cells exposed to purified histones (50 μg/ml, 1 h), chromogenic assay. B ) [Ca 2+ ] i staining in untreated (Ctrl)

    Article Snippet: The following reagents were used: anti-Ly6G antibody (clone RB6-8C5) and rat IgG2bκ isotype antibody (eBioscience), neutralizing anti-histone H4 antibody (clone BWA3; ref. ) and mouse IgG1κ isotype antibody (BioLegend), rmC5a (R & D Systems), purified histones from calf thymus (Sigma), histone H1 and histone H3 (Roche), and histone H4 (BPS Bioscience, San Diego, CA, USA).

    Techniques: Purification, Chromogenic Assay, Staining

    Administration of extracellular histones into airways induces severe disturbances in alveolar-capillary gas exchange. Purified histones (50 μg/g body weight) were administered i.t. to Sprague-Dawley rats with implanted carotid artery catheters.

    Journal: The FASEB Journal

    Article Title: Extracellular histones are essential effectors of C5aR- and C5L2-mediated tissue damage and inflammation in acute lung injury

    doi: 10.1096/fj.13-236380

    Figure Lengend Snippet: Administration of extracellular histones into airways induces severe disturbances in alveolar-capillary gas exchange. Purified histones (50 μg/g body weight) were administered i.t. to Sprague-Dawley rats with implanted carotid artery catheters.

    Article Snippet: The following reagents were used: anti-Ly6G antibody (clone RB6-8C5) and rat IgG2bκ isotype antibody (eBioscience), neutralizing anti-histone H4 antibody (clone BWA3; ref. ) and mouse IgG1κ isotype antibody (BioLegend), rmC5a (R & D Systems), purified histones from calf thymus (Sigma), histone H1 and histone H3 (Roche), and histone H4 (BPS Bioscience, San Diego, CA, USA).

    Techniques: Purification

    Protective effects of neutralization of extracellular histones during ALI. A ) Reduced alveolar permeability disturbances after C5a-ALI with treatment of neutralizing anti-H4 antibody (BWA3, 250 μg i.v. + 50 μg i.t.) or isotype control

    Journal: The FASEB Journal

    Article Title: Extracellular histones are essential effectors of C5aR- and C5L2-mediated tissue damage and inflammation in acute lung injury

    doi: 10.1096/fj.13-236380

    Figure Lengend Snippet: Protective effects of neutralization of extracellular histones during ALI. A ) Reduced alveolar permeability disturbances after C5a-ALI with treatment of neutralizing anti-H4 antibody (BWA3, 250 μg i.v. + 50 μg i.t.) or isotype control

    Article Snippet: The following reagents were used: anti-Ly6G antibody (clone RB6-8C5) and rat IgG2bκ isotype antibody (eBioscience), neutralizing anti-histone H4 antibody (clone BWA3; ref. ) and mouse IgG1κ isotype antibody (BioLegend), rmC5a (R & D Systems), purified histones from calf thymus (Sigma), histone H1 and histone H3 (Roche), and histone H4 (BPS Bioscience, San Diego, CA, USA).

    Techniques: Neutralization, Permeability

    Detection of extracellular histones during ALI in humans or mice. A ) Top panel: Western blotting of histone H4 in BALF from 2 humans with ALI/ARDS (40589; 50131) at sequential time points (4–21 d), a healthy volunteer (Ctrl BALF, negative control),

    Journal: The FASEB Journal

    Article Title: Extracellular histones are essential effectors of C5aR- and C5L2-mediated tissue damage and inflammation in acute lung injury

    doi: 10.1096/fj.13-236380

    Figure Lengend Snippet: Detection of extracellular histones during ALI in humans or mice. A ) Top panel: Western blotting of histone H4 in BALF from 2 humans with ALI/ARDS (40589; 50131) at sequential time points (4–21 d), a healthy volunteer (Ctrl BALF, negative control),

    Article Snippet: The following reagents were used: anti-Ly6G antibody (clone RB6-8C5) and rat IgG2bκ isotype antibody (eBioscience), neutralizing anti-histone H4 antibody (clone BWA3; ref. ) and mouse IgG1κ isotype antibody (BioLegend), rmC5a (R & D Systems), purified histones from calf thymus (Sigma), histone H1 and histone H3 (Roche), and histone H4 (BPS Bioscience, San Diego, CA, USA).

    Techniques: Mouse Assay, Western Blot, Negative Control

    Induction of acute inflammation in lungs by extracellular histones. A ) Disruption of alveolar permeability in C57BL/6J mice after intratracheal deposition of purified histones at different doses, 8 h; BALF albumin ELISA. B ) Time course for alveolar permeability

    Journal: The FASEB Journal

    Article Title: Extracellular histones are essential effectors of C5aR- and C5L2-mediated tissue damage and inflammation in acute lung injury

    doi: 10.1096/fj.13-236380

    Figure Lengend Snippet: Induction of acute inflammation in lungs by extracellular histones. A ) Disruption of alveolar permeability in C57BL/6J mice after intratracheal deposition of purified histones at different doses, 8 h; BALF albumin ELISA. B ) Time course for alveolar permeability

    Article Snippet: The following reagents were used: anti-Ly6G antibody (clone RB6-8C5) and rat IgG2bκ isotype antibody (eBioscience), neutralizing anti-histone H4 antibody (clone BWA3; ref. ) and mouse IgG1κ isotype antibody (BioLegend), rmC5a (R & D Systems), purified histones from calf thymus (Sigma), histone H1 and histone H3 (Roche), and histone H4 (BPS Bioscience, San Diego, CA, USA).

    Techniques: Permeability, Mouse Assay, Purification, Enzyme-linked Immunosorbent Assay

    Extracellular histones mediate lung consolidation and acute histopathology in lungs. A ) High-resolution MRI of lungs from C57BL/6J mice after PBS i.t. (sham treatment) or purified histones (20 μg/g body weight), 6 h. Arrowheads indicate signal-intense

    Journal: The FASEB Journal

    Article Title: Extracellular histones are essential effectors of C5aR- and C5L2-mediated tissue damage and inflammation in acute lung injury

    doi: 10.1096/fj.13-236380

    Figure Lengend Snippet: Extracellular histones mediate lung consolidation and acute histopathology in lungs. A ) High-resolution MRI of lungs from C57BL/6J mice after PBS i.t. (sham treatment) or purified histones (20 μg/g body weight), 6 h. Arrowheads indicate signal-intense

    Article Snippet: The following reagents were used: anti-Ly6G antibody (clone RB6-8C5) and rat IgG2bκ isotype antibody (eBioscience), neutralizing anti-histone H4 antibody (clone BWA3; ref. ) and mouse IgG1κ isotype antibody (BioLegend), rmC5a (R & D Systems), purified histones from calf thymus (Sigma), histone H1 and histone H3 (Roche), and histone H4 (BPS Bioscience, San Diego, CA, USA).

    Techniques: Histopathology, Magnetic Resonance Imaging, Mouse Assay, Purification

    Whole-body plethysmography following airway administration of histones. Sprague-Dawley rats received purified histones (50 μg/g body weight i.t.) or PBS i.t (sham treatment). Respiration was monitored before the intervention and at intervals during

    Journal: The FASEB Journal

    Article Title: Extracellular histones are essential effectors of C5aR- and C5L2-mediated tissue damage and inflammation in acute lung injury

    doi: 10.1096/fj.13-236380

    Figure Lengend Snippet: Whole-body plethysmography following airway administration of histones. Sprague-Dawley rats received purified histones (50 μg/g body weight i.t.) or PBS i.t (sham treatment). Respiration was monitored before the intervention and at intervals during

    Article Snippet: The following reagents were used: anti-Ly6G antibody (clone RB6-8C5) and rat IgG2bκ isotype antibody (eBioscience), neutralizing anti-histone H4 antibody (clone BWA3; ref. ) and mouse IgG1κ isotype antibody (BioLegend), rmC5a (R & D Systems), purified histones from calf thymus (Sigma), histone H1 and histone H3 (Roche), and histone H4 (BPS Bioscience, San Diego, CA, USA).

    Techniques: Purification

    CARM1 represses γ-globin expression. A , Western blot analysis of indicated protein levels in cell lysates from the scramble control (Scr), CARM1-KD1, and CARM1-KD2 Lys-562 cells. GAPDH and histone H4 were used as loading controls. Blots are representative of three independent experiments. B and C , quantitative real-time PCR analysis of CARM1 ( B ) and γ-globin ( C ) mRNA level in Scr, CARM1-KD1, and CARM1-KD2 Lys-562 cells normalized to β-actin mRNA. The results are shown as the mean ± S.D. from three independent experiments. Two-tailed Student's t -tests were used to compare means. **, p

    Journal: The Journal of Biological Chemistry

    Article Title: CARM1-mediated methylation of protein arginine methyltransferase 5 represses human γ-globin gene expression in erythroleukemia cells

    doi: 10.1074/jbc.RA118.004028

    Figure Lengend Snippet: CARM1 represses γ-globin expression. A , Western blot analysis of indicated protein levels in cell lysates from the scramble control (Scr), CARM1-KD1, and CARM1-KD2 Lys-562 cells. GAPDH and histone H4 were used as loading controls. Blots are representative of three independent experiments. B and C , quantitative real-time PCR analysis of CARM1 ( B ) and γ-globin ( C ) mRNA level in Scr, CARM1-KD1, and CARM1-KD2 Lys-562 cells normalized to β-actin mRNA. The results are shown as the mean ± S.D. from three independent experiments. Two-tailed Student's t -tests were used to compare means. **, p

    Article Snippet: The following antibodies were used for Western blotting: FLAG (Sigma-Aldrich), PRMT5 (Sigma-Aldrich), CARM1 (Cell Signaling Technology), GAPDH (MBL International), H4R3me2s (Abcam), H4R3me2a (Active Motif), H3R17me2a (Abcam), histone H4 (GenScript), and histone H3 (GenScript).

    Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Two Tailed Test

    CARM1 methylates PRMT5. A , SDS-PAGE analysis of purified recombinant PRMT5 (rPRMT5) from E. coli as the substrate for following in vitro methylation assays. B , SDS-PAGE analysis of purified recombinant GST-PRMT1 and GST control from E. coli ( top left ). SDS-PAGE analysis of free histones stained by Coomassie Brilliant Blue ( CBB ) ( middle left ). Western blot analysis of free histones from an in vitro methylation assay with H4R3me2a antibody ( bottom left ). Autoradiographic image from an in vitro methylation assay with GST-PRMT1 or GST control ( right ). C , SDS-PAGE analysis of purified recombinant GST-CARM1 and GST control from E. coli ( top left ). SDS-PAGE analysis of free histones stained by Coomassie Brilliant Blue ( CBB ) ( middle left ). Western blot analysis of free histones from an in vitro methylation assay with H3R17me2a antibody ( bottom left ). Autoradiographic image from an in vitro methylation assay with GST-CARM1 or GST control ( right ). A gray asterisk denotes the positive 3 H-labeled PRMT5 band. D , SDS-PAGE analysis of IgG control ( top left ) and of FLAG-PRMT5 immunoprecipitated from Lys-562 cells overexpressing FLAG-tagged PRMT5. SDS-PAGE analysis of free histones stained by Coomassie Brilliant Blue ( CBB ) ( middle left ). Western blot analysis of free histones from an in vitro methylation assay with H4R3me2s antibody ( bottom left ). Autoradiographic image from an in vitro methylation assay with FLAG-PRMT5 or IgG control ( right ). Black asterisks denote the fusion proteins in assays.

    Journal: The Journal of Biological Chemistry

    Article Title: CARM1-mediated methylation of protein arginine methyltransferase 5 represses human γ-globin gene expression in erythroleukemia cells

    doi: 10.1074/jbc.RA118.004028

    Figure Lengend Snippet: CARM1 methylates PRMT5. A , SDS-PAGE analysis of purified recombinant PRMT5 (rPRMT5) from E. coli as the substrate for following in vitro methylation assays. B , SDS-PAGE analysis of purified recombinant GST-PRMT1 and GST control from E. coli ( top left ). SDS-PAGE analysis of free histones stained by Coomassie Brilliant Blue ( CBB ) ( middle left ). Western blot analysis of free histones from an in vitro methylation assay with H4R3me2a antibody ( bottom left ). Autoradiographic image from an in vitro methylation assay with GST-PRMT1 or GST control ( right ). C , SDS-PAGE analysis of purified recombinant GST-CARM1 and GST control from E. coli ( top left ). SDS-PAGE analysis of free histones stained by Coomassie Brilliant Blue ( CBB ) ( middle left ). Western blot analysis of free histones from an in vitro methylation assay with H3R17me2a antibody ( bottom left ). Autoradiographic image from an in vitro methylation assay with GST-CARM1 or GST control ( right ). A gray asterisk denotes the positive 3 H-labeled PRMT5 band. D , SDS-PAGE analysis of IgG control ( top left ) and of FLAG-PRMT5 immunoprecipitated from Lys-562 cells overexpressing FLAG-tagged PRMT5. SDS-PAGE analysis of free histones stained by Coomassie Brilliant Blue ( CBB ) ( middle left ). Western blot analysis of free histones from an in vitro methylation assay with H4R3me2s antibody ( bottom left ). Autoradiographic image from an in vitro methylation assay with FLAG-PRMT5 or IgG control ( right ). Black asterisks denote the fusion proteins in assays.

    Article Snippet: The following antibodies were used for Western blotting: FLAG (Sigma-Aldrich), PRMT5 (Sigma-Aldrich), CARM1 (Cell Signaling Technology), GAPDH (MBL International), H4R3me2s (Abcam), H4R3me2a (Active Motif), H3R17me2a (Abcam), histone H4 (GenScript), and histone H3 (GenScript).

    Techniques: SDS Page, Purification, Recombinant, In Vitro, Methylation, Staining, Western Blot, Labeling, Immunoprecipitation

    Methylation of PRMT5 at Arg-505 is essential for its methyltransferase activity. A , Western blot analysis of extracts from Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ using FLAG and PRMT5 antibodies. GAPDH was used as a loading control. Blots are representative of three independent experiments. B, quantitative real-time PCR analysis of PRMT5 mRNA normalized to β-actin in Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ. The results are shown as the mean ± S.D. from three independent experiments. C , Western blot analysis of extracted histones from Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ using H4R3me2s antibody. Histone H4 was used as a loading control. Blots are representative of three independent experiments. D , ChIP analysis of H4R3me2s enrichment at the γ-promoter in Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ. The results are shown as the mean ± S.D. from three independent experiments. Two-tailed Student's t -tests were used to compare means. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: CARM1-mediated methylation of protein arginine methyltransferase 5 represses human γ-globin gene expression in erythroleukemia cells

    doi: 10.1074/jbc.RA118.004028

    Figure Lengend Snippet: Methylation of PRMT5 at Arg-505 is essential for its methyltransferase activity. A , Western blot analysis of extracts from Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ using FLAG and PRMT5 antibodies. GAPDH was used as a loading control. Blots are representative of three independent experiments. B, quantitative real-time PCR analysis of PRMT5 mRNA normalized to β-actin in Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ. The results are shown as the mean ± S.D. from three independent experiments. C , Western blot analysis of extracted histones from Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ using H4R3me2s antibody. Histone H4 was used as a loading control. Blots are representative of three independent experiments. D , ChIP analysis of H4R3me2s enrichment at the γ-promoter in Lys-562 cells containing vector or overexpressing PRMT5-WT, PRMT5-R505A, PRMT5-R505K, or PRMT5Δ. The results are shown as the mean ± S.D. from three independent experiments. Two-tailed Student's t -tests were used to compare means. *, p

    Article Snippet: The following antibodies were used for Western blotting: FLAG (Sigma-Aldrich), PRMT5 (Sigma-Aldrich), CARM1 (Cell Signaling Technology), GAPDH (MBL International), H4R3me2s (Abcam), H4R3me2a (Active Motif), H3R17me2a (Abcam), histone H4 (GenScript), and histone H3 (GenScript).

    Techniques: Methylation, Activity Assay, Western Blot, Plasmid Preparation, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Two Tailed Test

    Cross-linking of E7 peptides to CDK2/cyclin A complex. (A) HPV16 E7 (1.8 μM) and E7-derived peptides (50 μM) stimulate CDK2/cyclin A histone kinase activity. (B) Benzo-CYS-9-48-bio and benzo-NH 2 -9-48-bio peptides associate with cyclin A/CDK2 complex following UV irradiation. Histone H1 is not cross-linked under identical conditions. (C) CDK2/cyclin A (0.1 μM) and benzo-CYS-9-48-bio (0.5 μM) were with or without competitor E7 or E7 peptide. Both full-length 16E7 and the 9-48 16E7 peptide compete with the benzo-CYS-9-48-bio peptide for binding to the cyclin A/CDK2 complex. These results suggest that labeling of the CDK2/cyclin A complex by benzo-CYS-9-48-bio is specific and is not due to indiscriminate labeling of the complex. (D) Immunoprecipitation (IP) followed by Western blotting for incorporation of biotin-containing peptide into immunoprecipitated target protein(s) demonstrates that biotin-reactive bands in Western blots are cyclin A and CDK2 and that reactivity of these bands with benzo-CYS-9-48-bio peptide is dependent upon UV. (E) Cross-linking of benzophenone-containing peptides to increasing amounts of GST, cyclin A, or CDK2 demonstrates a strong preference of the peptide for cyclin A.

    Journal: Journal of Virology

    Article Title: Direct Activation of Cyclin-Dependent Kinase 2 by Human Papillomavirus E7

    doi: 10.1128/JVI.77.19.10566-10574.2003

    Figure Lengend Snippet: Cross-linking of E7 peptides to CDK2/cyclin A complex. (A) HPV16 E7 (1.8 μM) and E7-derived peptides (50 μM) stimulate CDK2/cyclin A histone kinase activity. (B) Benzo-CYS-9-48-bio and benzo-NH 2 -9-48-bio peptides associate with cyclin A/CDK2 complex following UV irradiation. Histone H1 is not cross-linked under identical conditions. (C) CDK2/cyclin A (0.1 μM) and benzo-CYS-9-48-bio (0.5 μM) were with or without competitor E7 or E7 peptide. Both full-length 16E7 and the 9-48 16E7 peptide compete with the benzo-CYS-9-48-bio peptide for binding to the cyclin A/CDK2 complex. These results suggest that labeling of the CDK2/cyclin A complex by benzo-CYS-9-48-bio is specific and is not due to indiscriminate labeling of the complex. (D) Immunoprecipitation (IP) followed by Western blotting for incorporation of biotin-containing peptide into immunoprecipitated target protein(s) demonstrates that biotin-reactive bands in Western blots are cyclin A and CDK2 and that reactivity of these bands with benzo-CYS-9-48-bio peptide is dependent upon UV. (E) Cross-linking of benzophenone-containing peptides to increasing amounts of GST, cyclin A, or CDK2 demonstrates a strong preference of the peptide for cyclin A.

    Article Snippet: The role of histone H1 in chromatin condensation and transcriptional repression.

    Techniques: Derivative Assay, Activity Assay, Irradiation, Binding Assay, Labeling, Immunoprecipitation, Western Blot

    Addition of increasing amounts of GST-16E7 to CDK2 purified from insect cells or from bacteria results in a dose-dependent increase in kinase activity. Autoradiograph (A) demonstrates E7-dependent increase in phosphorylation of histone H1 by CDK2/cyclin A. Quantification of the experiment (B) plots PhosphorImager counts versus amount of GST-16E7 added. Results indicate that E7 affect peaks at 640 ng of E7 (or approximately 0.6 μM E7). CDK2/cyclin A reconstituted from bacterially expressed proteins is also activated by purified 16E7 (C).

    Journal: Journal of Virology

    Article Title: Direct Activation of Cyclin-Dependent Kinase 2 by Human Papillomavirus E7

    doi: 10.1128/JVI.77.19.10566-10574.2003

    Figure Lengend Snippet: Addition of increasing amounts of GST-16E7 to CDK2 purified from insect cells or from bacteria results in a dose-dependent increase in kinase activity. Autoradiograph (A) demonstrates E7-dependent increase in phosphorylation of histone H1 by CDK2/cyclin A. Quantification of the experiment (B) plots PhosphorImager counts versus amount of GST-16E7 added. Results indicate that E7 affect peaks at 640 ng of E7 (or approximately 0.6 μM E7). CDK2/cyclin A reconstituted from bacterially expressed proteins is also activated by purified 16E7 (C).

    Article Snippet: The role of histone H1 in chromatin condensation and transcriptional repression.

    Techniques: Purification, Activity Assay, Autoradiography

    GST-16E7 activates purified cyclin E/CDK2 and cyclin A/CDK2 complexes. Autoradiographs demonstrate phosphorylation of histone H1 and Rb (pRb) by CDK2/cyclin E (A) and CDK2/cyclin A (B). (A) E7 potently upregulates CDK2/cyclin E histone H1 kinase activity while having modest effects on Rb kinase activity. Addition of p27 eliminates this effect and reduces activity to below baseline levels. (B) CDK2/cyclin A histone kinase activity is also dramatically upregulated by E7, and the effect is negated by addition of p27.

    Journal: Journal of Virology

    Article Title: Direct Activation of Cyclin-Dependent Kinase 2 by Human Papillomavirus E7

    doi: 10.1128/JVI.77.19.10566-10574.2003

    Figure Lengend Snippet: GST-16E7 activates purified cyclin E/CDK2 and cyclin A/CDK2 complexes. Autoradiographs demonstrate phosphorylation of histone H1 and Rb (pRb) by CDK2/cyclin E (A) and CDK2/cyclin A (B). (A) E7 potently upregulates CDK2/cyclin E histone H1 kinase activity while having modest effects on Rb kinase activity. Addition of p27 eliminates this effect and reduces activity to below baseline levels. (B) CDK2/cyclin A histone kinase activity is also dramatically upregulated by E7, and the effect is negated by addition of p27.

    Article Snippet: The role of histone H1 in chromatin condensation and transcriptional repression.

    Techniques: Purification, Activity Assay

    An ACLY knockdown does not recapitulate the phenotype of ACLY inhibitor-treated MDMs. (A–D) Q-PCR analysis of mRNA expression of ALOX15, CCL17, F13A1, and ACLY in MDMs transfected with 50 nM ACLY siRNA for 96 h prior to 24-h treatment with 20 ng/mL IL-4. (E–G) Western blot analysis of ACLY protein expression (E) , assay of enzymatic activity (F) , and Western blot analysis of histone H3 acetylation at K27 and K14 (G) 96 h post-transfection with ACLY siRNA. ** p

    Journal: Frontiers in Immunology

    Article Title: Polarization of Human Macrophages by Interleukin-4 Does Not Require ATP-Citrate Lyase

    doi: 10.3389/fimmu.2018.02858

    Figure Lengend Snippet: An ACLY knockdown does not recapitulate the phenotype of ACLY inhibitor-treated MDMs. (A–D) Q-PCR analysis of mRNA expression of ALOX15, CCL17, F13A1, and ACLY in MDMs transfected with 50 nM ACLY siRNA for 96 h prior to 24-h treatment with 20 ng/mL IL-4. (E–G) Western blot analysis of ACLY protein expression (E) , assay of enzymatic activity (F) , and Western blot analysis of histone H3 acetylation at K27 and K14 (G) 96 h post-transfection with ACLY siRNA. ** p

    Article Snippet: Following primary antibodies were used: pSTAT6 (Y641) (cat. no. #9361), STAT6 (clone D3H4) (cat. no. #5397), pSTAT3 (Y705) (cat. no. #9131), STAT3 (clone 124H6) (cat. no. #9139), pACLY (S454) (cat. no. #4331), histone H3 acK14 (clone D4B9) (cat. no. #7627), histone H3 acK23 (clone D6Y7M) (cat. no. #14932), DDK tag (clone 9A3) (cat. no. #8146) (all Cell Signaling Technology), ACLY (cat. no. 15421-1-AP, Proteintech), histone H3 acK9 (clone Y28) (cat. no. 04-1003, Merck Millipore), histone H3 acK27 (cat. no. C15410174, Diagenode), histone H3 (cat. no. 07-690, Merck Millipore), nucleolin (cat. no. sc-13057, Santa-Cruz).

    Techniques: Polymerase Chain Reaction, Expressing, Transfection, Western Blot, Activity Assay

    ACLY inhibitors suppress IL-4-induced gene expression in ACLY knockout THP-1 cells. (A,B) Western blot analysis of ACLY protein expression (A) and growth curves (B) of control and ACLY knockout (ACLY KO) THP-1 cells. (C) Western blot analysis of histone H3 acetylation at K9, K23, K27, and K14 in control or ACLY KO THP-1 macrophages. (D,E) Q-PCR analysis of IL-4—induced mRNA expression of CCL13 and F13A1 in control or ACLY KO THP-1 macrophages. (F) Western blot analysis of STAT6 phosphorylation in control or ACLY KO THP-1 macrophages. (G,H) Q-PCR analysis of mRNA expression of CCL13 and F13A1 in ACLY KO THP-1 macrophages stimulated with IL-4 in the presence of BMS 303141, SB 204990, MEDICA16 or HC. * p

    Journal: Frontiers in Immunology

    Article Title: Polarization of Human Macrophages by Interleukin-4 Does Not Require ATP-Citrate Lyase

    doi: 10.3389/fimmu.2018.02858

    Figure Lengend Snippet: ACLY inhibitors suppress IL-4-induced gene expression in ACLY knockout THP-1 cells. (A,B) Western blot analysis of ACLY protein expression (A) and growth curves (B) of control and ACLY knockout (ACLY KO) THP-1 cells. (C) Western blot analysis of histone H3 acetylation at K9, K23, K27, and K14 in control or ACLY KO THP-1 macrophages. (D,E) Q-PCR analysis of IL-4—induced mRNA expression of CCL13 and F13A1 in control or ACLY KO THP-1 macrophages. (F) Western blot analysis of STAT6 phosphorylation in control or ACLY KO THP-1 macrophages. (G,H) Q-PCR analysis of mRNA expression of CCL13 and F13A1 in ACLY KO THP-1 macrophages stimulated with IL-4 in the presence of BMS 303141, SB 204990, MEDICA16 or HC. * p

    Article Snippet: Following primary antibodies were used: pSTAT6 (Y641) (cat. no. #9361), STAT6 (clone D3H4) (cat. no. #5397), pSTAT3 (Y705) (cat. no. #9131), STAT3 (clone 124H6) (cat. no. #9139), pACLY (S454) (cat. no. #4331), histone H3 acK14 (clone D4B9) (cat. no. #7627), histone H3 acK23 (clone D6Y7M) (cat. no. #14932), DDK tag (clone 9A3) (cat. no. #8146) (all Cell Signaling Technology), ACLY (cat. no. 15421-1-AP, Proteintech), histone H3 acK9 (clone Y28) (cat. no. 04-1003, Merck Millipore), histone H3 acK27 (cat. no. C15410174, Diagenode), histone H3 (cat. no. 07-690, Merck Millipore), nucleolin (cat. no. sc-13057, Santa-Cruz).

    Techniques: Expressing, Knock-Out, Western Blot, Polymerase Chain Reaction

    Effects of ACLY inhibitors on acetyl-CoA levels, histone acetylation, and IL-4-induced STAT6 and STAT3 activation in human MDMs. (A) LC-MS analysis of acetyl-CoA levels in MDMs treated with 5 μM BMS 303141 and 10 μM SB 204990 for 24 h. (B) Western blot analysis of histone H3 acetylation at K14 and K27 in MDMs treated with indicated concentrations of BMS 303141 and SB 204990 for 24 h. (C–F) Q-PCR analysis of mRNA expression of ALOX15 and CCL17 in MDMs pre-treated with acetate, octanoate and TOFA for 1 h prior to treatments with SB 204490, hydroxycitrate, and IL-4 for 24 h. (G,H) Q-PCR analysis of mRNA expression of ALOX15 in MDMs pre-treated with indicated concentrations of BTA (G) or CTPi (H) for 1 h prior to treatment with IL-4 for 24 h. (I) Western blot analysis of STAT3 and STAT6 tyrosine phosphorylation in MDMs pre-treated with ACLY inhibitors 1 h prior to 0.5 h-treatment with IL-4. * p

    Journal: Frontiers in Immunology

    Article Title: Polarization of Human Macrophages by Interleukin-4 Does Not Require ATP-Citrate Lyase

    doi: 10.3389/fimmu.2018.02858

    Figure Lengend Snippet: Effects of ACLY inhibitors on acetyl-CoA levels, histone acetylation, and IL-4-induced STAT6 and STAT3 activation in human MDMs. (A) LC-MS analysis of acetyl-CoA levels in MDMs treated with 5 μM BMS 303141 and 10 μM SB 204990 for 24 h. (B) Western blot analysis of histone H3 acetylation at K14 and K27 in MDMs treated with indicated concentrations of BMS 303141 and SB 204990 for 24 h. (C–F) Q-PCR analysis of mRNA expression of ALOX15 and CCL17 in MDMs pre-treated with acetate, octanoate and TOFA for 1 h prior to treatments with SB 204490, hydroxycitrate, and IL-4 for 24 h. (G,H) Q-PCR analysis of mRNA expression of ALOX15 in MDMs pre-treated with indicated concentrations of BTA (G) or CTPi (H) for 1 h prior to treatment with IL-4 for 24 h. (I) Western blot analysis of STAT3 and STAT6 tyrosine phosphorylation in MDMs pre-treated with ACLY inhibitors 1 h prior to 0.5 h-treatment with IL-4. * p

    Article Snippet: Following primary antibodies were used: pSTAT6 (Y641) (cat. no. #9361), STAT6 (clone D3H4) (cat. no. #5397), pSTAT3 (Y705) (cat. no. #9131), STAT3 (clone 124H6) (cat. no. #9139), pACLY (S454) (cat. no. #4331), histone H3 acK14 (clone D4B9) (cat. no. #7627), histone H3 acK23 (clone D6Y7M) (cat. no. #14932), DDK tag (clone 9A3) (cat. no. #8146) (all Cell Signaling Technology), ACLY (cat. no. 15421-1-AP, Proteintech), histone H3 acK9 (clone Y28) (cat. no. 04-1003, Merck Millipore), histone H3 acK27 (cat. no. C15410174, Diagenode), histone H3 (cat. no. 07-690, Merck Millipore), nucleolin (cat. no. sc-13057, Santa-Cruz).

    Techniques: Activation Assay, Liquid Chromatography with Mass Spectroscopy, Western Blot, Polymerase Chain Reaction, Expressing

    IL-4 does not induce ACLY phosphorylation in human MDMs. (A–C) Western blot analysis of ACLY and Akt phosphorylation (A) , nuclear ACLY levels (B) , and histone H3 acetylation at K27 and K14 (C) in MDMs treated with 20 ng/mL IL-4 for indicated times. Data represent mean values ± SE of 3–4 independent experiments. * p

    Journal: Frontiers in Immunology

    Article Title: Polarization of Human Macrophages by Interleukin-4 Does Not Require ATP-Citrate Lyase

    doi: 10.3389/fimmu.2018.02858

    Figure Lengend Snippet: IL-4 does not induce ACLY phosphorylation in human MDMs. (A–C) Western blot analysis of ACLY and Akt phosphorylation (A) , nuclear ACLY levels (B) , and histone H3 acetylation at K27 and K14 (C) in MDMs treated with 20 ng/mL IL-4 for indicated times. Data represent mean values ± SE of 3–4 independent experiments. * p

    Article Snippet: Following primary antibodies were used: pSTAT6 (Y641) (cat. no. #9361), STAT6 (clone D3H4) (cat. no. #5397), pSTAT3 (Y705) (cat. no. #9131), STAT3 (clone 124H6) (cat. no. #9139), pACLY (S454) (cat. no. #4331), histone H3 acK14 (clone D4B9) (cat. no. #7627), histone H3 acK23 (clone D6Y7M) (cat. no. #14932), DDK tag (clone 9A3) (cat. no. #8146) (all Cell Signaling Technology), ACLY (cat. no. 15421-1-AP, Proteintech), histone H3 acK9 (clone Y28) (cat. no. 04-1003, Merck Millipore), histone H3 acK27 (cat. no. C15410174, Diagenode), histone H3 (cat. no. 07-690, Merck Millipore), nucleolin (cat. no. sc-13057, Santa-Cruz).

    Techniques: Western Blot

    Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

    Journal: Nature

    Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

    doi: 10.1038/s41586-019-1167-6

    Figure Lengend Snippet: Therapeutic disruption of the histone H4-plasma membrane interaction stabilizes atherosclerotic lesions.

    Article Snippet: SMCs were incubated with indicated amounts of isolated NETs, histone H4 (Biomol), or equimolecular amounts of histone H4 fragments (Pepscan).

    Techniques:

    Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

    Journal: Nature

    Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

    doi: 10.1038/s41586-019-1167-6

    Figure Lengend Snippet: Neutralization of histone H4 stabilizes atherosclerotic lesions. a , Experimental scheme. b – i , Quantification of lesion characteristics of the carotid artery. Displayed are lesion volume ( b ), lesion size ( c ), fibrous cap (FC) thickness ( d ), necrotic core area ( e ), collagen area ( f ), macrophage area (CD68 + , g ), SMA + MYH11 + cells ( h ) and SMA − MYH11 + cells ( i ). n = 14 mice (ctrl IgG) except for ( h , i ) n = 11 mice; n = 15 mice (anti-histone H4) except for ( h , i ) n = 12 mice. Two-sided unpaired t -test. j – p , Quantification of lesion characteristics on the brachiocephalic artery. Displayed are lesion size ( j ), fibrous cap (FC) thickness ( k ), necrotic core area ( l ), collagen area ( m ), SMCs (SMA + , n ), macrophages (CD68 + , o ) and overall vulnerability ( p ). Two-sided unpaired t -test, n = 12 mice (ctrl IgG) or 10 mice (anti-histone H4). Data are mean ± s.d.

    Article Snippet: SMCs were incubated with indicated amounts of isolated NETs, histone H4 (Biomol), or equimolecular amounts of histone H4 fragments (Pepscan).

    Techniques: Neutralization, Mouse Assay

    NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

    Journal: Nature

    Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

    doi: 10.1038/s41586-019-1167-6

    Figure Lengend Snippet: NET-derived histone H4 induces cell toxicity. a – c , Analysis of cell death (propidium iodide uptake). a , NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b , NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c , SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t -test, * P = 0.029; ** P = 3.847 × 10 −5 ; *** P = 8.775 × 10 −6 . d , Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 μm. e , Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, * P = 0.02; ** P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.

    Article Snippet: SMCs were incubated with indicated amounts of isolated NETs, histone H4 (Biomol), or equimolecular amounts of histone H4 fragments (Pepscan).

    Techniques: Derivative Assay, Incubation, Recombinant, Immunofluorescence

    NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

    Journal: Nature

    Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

    doi: 10.1038/s41586-019-1167-6

    Figure Lengend Snippet: NET-derived histone H4 interaction with cell membranes is surface charge dependent and induces a lytic cell death. a , SMCs were pre-incubated with indicated inhibitors before NET treatment. Cell death was assessed by PI uptake. n = 24 fields, except TLR4, n = 23 fields. One-way ANOVA with Dunnet’s correction, P = 0.729 (TLR1/2), P = 0.999 (TLR3), P = 0.995 (TLR4). All conditions were compared against control (ctrl). b , Representative high-resolution confocal microscopy images were used to visualize cell membrane (lectin, white), histone H4 (magenta) and DNA (DAPI, cyan) in a SMC (S) and neutrophil (N) co-culture. Dashed lines indicate cross-section views represented in . c . d , e , SMCs were incubated with NETs. d, Extracellular ATP. n = 3 biological replicates (crtl), n = 6 biological replicates (NETs). Twosided Mann-Whitney test. e , Flow cytometry analysis of cell size. n = 9 biological replicates. Two-sided unpaired t -test. f , g , SMCs were incubated with histone H4. f , Extracellular ATP. n = 5 biological replicates. Two-sided Mann-Whitney test. g , Time-lapse microscopy images were used to measure SMC area before and after incubation with histone H4. n = 9 cells. Two-sided paired t -test. h , Analysis of the ζ potential of SMCs incubated with oleylamine or cholesterol sulfate (chl sulfate). n = 9 biological replicates (ctrl), n = 8 biological replicates (oleylamine), n = 6 biological replicates (chl sulfate). Two-sided Mann-Whitney test. i , j , SMCs were incubated with recombinant histone H4 after preincubation with oleylamine or cholesterol sulfate (chl sulfate). i , Confocal microscopy was used to detect histone H4 and plasma cell membrane (phalloidin). Peptide-membrane interaction quantified as the ratio of histone H4-fragment signal and plasma membrane area. n = 10 cells (ctrl), n = 20 cells (histone H4, –), n = 20 cells (oleylamine), n = 25 cells (chl sulfate). One-way ANOVA with Dunnet’s correction. ** P = 0.007; *** P = 0.0001 vs ctrl. j , Quantification of PI incorporation. n = 54 fields, n = 8 fields, n = 10 fields, n = 34 fields, n = 21 fields and n = 19 fields for each condition represented. One-way ANOVA with Tukey’s correction, * P = 0.001; ** P = 0.004. Data are mean ± s.d. Fig. 3j

    Article Snippet: SMCs were incubated with indicated amounts of isolated NETs, histone H4 (Biomol), or equimolecular amounts of histone H4 fragments (Pepscan).

    Techniques: Derivative Assay, Incubation, Confocal Microscopy, Co-Culture Assay, MANN-WHITNEY, Flow Cytometry, Cytometry, Time-lapse Microscopy, Recombinant

    NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

    Journal: Nature

    Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

    doi: 10.1038/s41586-019-1167-6

    Figure Lengend Snippet: NET-derived histone H4 induces SMC lysis and exacerbates plaque instability.

    Article Snippet: SMCs were incubated with indicated amounts of isolated NETs, histone H4 (Biomol), or equimolecular amounts of histone H4 fragments (Pepscan).

    Techniques: Derivative Assay, Lysis

    Membrane-pore-forming activity of histone H4.

    Journal: Nature

    Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

    doi: 10.1038/s41586-019-1167-6

    Figure Lengend Snippet: Membrane-pore-forming activity of histone H4.

    Article Snippet: SMCs were incubated with indicated amounts of isolated NETs, histone H4 (Biomol), or equimolecular amounts of histone H4 fragments (Pepscan).

    Techniques: Activity Assay

    Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

    Journal: Nature

    Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

    doi: 10.1038/s41586-019-1167-6

    Figure Lengend Snippet: Activated SMCs induce neutrophil chemotaxis and induce NET-mediated SMC death. a , Neutrophil displacement in gradient of supernatant obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 20 neutrophils (ctrl), n = 18 neutrophils (PDGF-BB). Two-way ANOVA. P = 1 × 10 −15 (ctrl vs PDGF-BB). MSD, mean square displacement. b, Neutrophils transmigrated towards supernatants obtained from PDGF-BB-activated or resting SMCs (ctrl). n = 14 replicates (ctrl), n = 11 replicates (PDGF-BB). Two-sided unpaired t -test. c , d , Multiplex ELISA of indicated growth factors and cytokines ( c ) and chemokines ( d ) in cell-free supernatants from SMCs treated with PDGF-BB or vehicle. n = 9 replicates (IL-6, CXCL12), n = 10 replicates (CXCL1, CCL5). Two-sided paired t -test. e , Pearson correlation between neutrophils and intimal CCL7 in mouse advanced atherosclerotic lesions, n = 28 sections. Dotted line represents 95% confidence interval. f , Representative micrographs of mouse atherosclerotic lesions showing SMCs (SMA, green), nuclei (blue), dead cells (TUNEL, red), and NETs (citrullinated histone H3, white). Dashed lines indicate cross-section views. Scale bar, 20 μm. Close-ups represent xz (left) and yz (right) cross-sections. Scale bar, 4 μm. Orange arrows indicate points of interactions between dead SMCs and NETs. g , Micrographs of mouse atherosclerotic lesions showing SMCs (MYH11, white), nuclei (blue), dead cells (TUNEL, red), and MPO (green). Yellow arrows indicate points of interactions between dead SMCs and NETs. Asterisks indicate intact MPO + cells. h – j , Advanced atherosclerotic lesions in the carotid artery were stained with antibodies to Ly6G, CD68, myeloperoxidase (MPO), and citrullinated H3 (citH3) and counterstained with DAPI. h , Representative images. Scale bar, 50 μm. i , Pie chart showing distribution of macrophage extracellular traps (METs, 1.86%), NETs (80.05%), and extracellular trap DNA (18.09%) based on marker analysis defined underneath, n = 35 sections from 8 mice. j , Extracellular trap DNA structures in carotid artery sections from neutropenic mice (anti-Ly6G, n = 13 sections), mice with intact white blood cell count (vehicle treated, n = 96 sections), or neutrophilic mice (AMD3100, n = 57 sections). Two-sided unpaired t -test. k , Percentage of viable SMCs after exposure to PMA-induced NETs isolated from indicated number of neutrophils. n = 16 biological samples (0, 2.75 × 10 6 neutrophils), n = 13 biological samples (0.27510 6 , 0.55 × 10 6 , 1.375 × 10 6 , 4.125 × 10 6 neutrophils), n = 11 biological samples (5.5 × 10 6 neutrophils). l , Cell death of SMCs incubated with NETs isolated from neutrophils treated with recombinant CCL7. n = 67 fields (−), n = 72 fields (+). Two-sided unpaired t -test, **** P = 0.000002. Data are mean ± s.d. MPO, myeloperoxidase; ND, not detected.

    Article Snippet: SMCs were incubated with indicated amounts of isolated NETs, histone H4 (Biomol), or equimolecular amounts of histone H4 fragments (Pepscan).

    Techniques: Chemotaxis Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Marker, Mouse Assay, Cell Counting, Isolation, Incubation, Recombinant

    Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

    Journal: Nature

    Article Title: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death

    doi: 10.1038/s41586-019-1167-6

    Figure Lengend Snippet: Membrane pore-forming activity of histone H4. a , Scanning electron micrographs of SMCs incubated with recombinant histone H4 or vehicle. b , Machine learning screen of full-length sequence histone H4 predicts potent membrane activity at the N terminus (residues 1–24 highlighted in blue). c , SAXS data demonstrates that N-terminal domain of histone H4 induces negative Gaussian curvature (NGC) in cell membranes at the indicated peptide:lipid (P/L) ratios. The histone H4 N terminus was incubated with indicated membrane compositions and the resulting structures were measured with SAXS. The peptide induced Pn3m cubic phases, which are rich in NGC, and are indicative of membrane permeation. d , SMCs were incubated with biotinylated histone H4 fragments (1–24: N terminus; 25–68: α-helix; 69–102: C terminus). Confocal microscopy was used to detect histone H4 fragments and plasma cell membrane. Peptide-membrane interaction was quantified as the ratio of histone H4 fragment signal and plasma membrane area. n = 44 cells (1–24), n = 28 cells (25–68), n = 33 cells (69–102). One-way ANOVA with Tukey’s correction; * P = 0.049; ** P = 4 × 10 −14 . e , PI incorporation in SMCs treated with histone H4 fragments or the full-length protein. n = 19 fields (ctrl), n = 24 fields (histone H4), n = 24 fields (1–24), n = 21 fields (25–68), n = 19 fields (69–102). One-way ANOVA with Dunnet’s correction; * P = 0.005; ** P = 0.0001 vs control. f , Histone H4 was preincubated with HIPe or vehicle and added to SMCs. Confocal microscopy was used to visualize interaction of histone H4 (green) with plasma cell membrane (phalloidin, red). n = 20 cells (ctrl), n = 17 cells (histone H4), n = 15 cells (histone H4+HIPe). One-way ANOVA with Tukey’s correction; * P = 9.243 × 10 −7 ; ** P = 6.239 × 10 −9 . Scale bar, 20 μm. g , Atomic force microscopy studies of lipid membranes treated with the indicated histone H4:HIPe ratio. Scale bar, 1 μm. Membrane disruption was quantified as membrane roughness. n = 13 membranes (ctrl), n = 3 membranes (1:0), n = 3 (1:1). Kruskal-Wallis test with Dunn’s correction. h , Live scanning ion conductance microscopy of SMCs. Images represent the plasma membrane before and after incubation with histone H4 and HIPe. i , PI incorporation in SMCs treated with recombinant histone H4 in the presence or absence of HIPe. n = 33 fields (ctrl), n = 12 fields (histone H4), n = 11 fields (histone H4 + HIPe). One-way ANOVA with Tukey’s correction; * P = 0.001; ** P = 8.844 × 10 −6 . Data are mean ± s.d.

    Article Snippet: SMCs were incubated with indicated amounts of isolated NETs, histone H4 (Biomol), or equimolecular amounts of histone H4 fragments (Pepscan).

    Techniques: Activity Assay, Incubation, Recombinant, Sequencing, Confocal Microscopy, Microscopy

    PITX2 binds to  SLC13A3  upstream elements. ChIP was used as template for standard PCR. Antibodies against PITX2 and acetyl-histone H3 (Lys9) (positive control for transcriptionally active chromatin) yielded template from which fragments of the SLC13A3

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: PITX2 Is Involved in Stress Response in Cultured Human Trabecular Meshwork Cells through Regulation of SLC13A3

    doi: 10.1167/iovs.10-6967

    Figure Lengend Snippet: PITX2 binds to SLC13A3 upstream elements. ChIP was used as template for standard PCR. Antibodies against PITX2 and acetyl-histone H3 (Lys9) (positive control for transcriptionally active chromatin) yielded template from which fragments of the SLC13A3

    Article Snippet: The ChIP reactions were set up by adding protein G magnetic beads, ChIP buffer 1, 6.3 μg sheared chromatin (∼300–1000 bp), protease inhibitor cocktail (P8340; Sigma-Aldrich Canada Ltd., Oakville, ON, Canada), and 2 μg of anti-PITX2 antibody (H00005308-M01; Abnova Corporation, Taipei, Taiwan), normal rabbit IgG antibody (10500C; Invitrogen), or anti-acetyl-histone H3 antibody (Lys9; 9671; Cell Signaling Technology, Inc., Danvers, MA).

    Techniques: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Positive Control