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  • 99
    Cell Signaling Technology Inc parp
    BCR-ABL deregulates miR-126 biogenesis (a,b) pri-miR-126 (n=8 biologically independent samples) (a) and pre-miR-126 (n=8 biologically independent samples) (b) expression levels, as assessed by QPCR, in the indicated human normal and CML cell populations. (c) BCR-ABL and SPRED1 staining in CML CD34 + cells by immunofluorescence (IF). (d) Immunoprecipitation (IP) with anti-SPRED1 followed by immunoblotting (IB) with anti-SPRED1 and anti-phosphotyrosine (p-Tyr) antibodies (left) and an in vitro kinase assay (right), as performed by IP with anti-c-Abl or anti-normal mouse IgG as control and immunoblotting with anti-SPRED1, in lysates of K562 cells treated with none, DMSO (vehicle) or NIL. (e) IP with <t>anti-RAN</t> followed by IB with anti-SPRED1 and anti-RAN antibodies in lysates of K562 cells. (f) SPRED1 and RAN staining by IF in K562 cells treated with none, DMSO or NIL. (g) SPRED1, RAN, RCC1 and Exp-5 expression in cytoplasmic (Cyt) and nuclear (Nu) fractions from K562 cells, treated with DMSO or NIL, as assessed by IB. Densitometric quantification of selected bands is shown (normalized to the actin loading control for total and Cyt lysates or to the <t>PARP</t> loading control for Nu lysates). (h) IP with anti-RAN followed by IB with anti-SPRED1, RAN, Exp-5 and RCC1 antibodies in lysates of K562 cells, CML CD34 + cells, and normal CD34+ cells treated with DMSO or NIL. Densitometric quantification of selected bands is shown (normalized to the actin loading control). (i) Mature, pri- and pre-miR-126 expression, as assessed by QPCR, in K562 and CML CD34 + cells treated with DMSO or NIL (n=3 independent experiments for K562 and 3 independent samples for CML cells). (j) IP with anti-RAN followed by IB with anti-SPRED1, Exp-5, RCC1 and RAN antibodies in lysates of K562 cells without or with washing-off of NIL. Densitometric quantification of selected bands is shown (normalized to the actin loading control). (k–m) Mature and pri-miR-126 expression as assessed by QPCR (n=3 independent experiments) (k), miR-126 staining (l), and mature and pre-miR-126 levels as assessed by Northern blotting (m) in K562 cells with or without washing-off of NIL. (n) IP with anti-RAN followed by IB with anti-Exp-5, RCC1 and RAN antibodies in lysates of K562 and CML CD34 + . Comparison between groups was performed by two-tailed, unpaired Student’s t -test. P values ≤0.05 were considered significant. Results shown represent mean ± SEM. * p ≤ 0.05, ** p
    Parp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 11597 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc cleaved parp
    BCR-ABL deregulates miR-126 biogenesis (a,b) pri-miR-126 (n=8 biologically independent samples) (a) and pre-miR-126 (n=8 biologically independent samples) (b) expression levels, as assessed by QPCR, in the indicated human normal and CML cell populations. (c) BCR-ABL and SPRED1 staining in CML CD34 + cells by immunofluorescence (IF). (d) Immunoprecipitation (IP) with anti-SPRED1 followed by immunoblotting (IB) with anti-SPRED1 and anti-phosphotyrosine (p-Tyr) antibodies (left) and an in vitro kinase assay (right), as performed by IP with anti-c-Abl or anti-normal mouse IgG as control and immunoblotting with anti-SPRED1, in lysates of K562 cells treated with none, DMSO (vehicle) or NIL. (e) IP with <t>anti-RAN</t> followed by IB with anti-SPRED1 and anti-RAN antibodies in lysates of K562 cells. (f) SPRED1 and RAN staining by IF in K562 cells treated with none, DMSO or NIL. (g) SPRED1, RAN, RCC1 and Exp-5 expression in cytoplasmic (Cyt) and nuclear (Nu) fractions from K562 cells, treated with DMSO or NIL, as assessed by IB. Densitometric quantification of selected bands is shown (normalized to the actin loading control for total and Cyt lysates or to the <t>PARP</t> loading control for Nu lysates). (h) IP with anti-RAN followed by IB with anti-SPRED1, RAN, Exp-5 and RCC1 antibodies in lysates of K562 cells, CML CD34 + cells, and normal CD34+ cells treated with DMSO or NIL. Densitometric quantification of selected bands is shown (normalized to the actin loading control). (i) Mature, pri- and pre-miR-126 expression, as assessed by QPCR, in K562 and CML CD34 + cells treated with DMSO or NIL (n=3 independent experiments for K562 and 3 independent samples for CML cells). (j) IP with anti-RAN followed by IB with anti-SPRED1, Exp-5, RCC1 and RAN antibodies in lysates of K562 cells without or with washing-off of NIL. Densitometric quantification of selected bands is shown (normalized to the actin loading control). (k–m) Mature and pri-miR-126 expression as assessed by QPCR (n=3 independent experiments) (k), miR-126 staining (l), and mature and pre-miR-126 levels as assessed by Northern blotting (m) in K562 cells with or without washing-off of NIL. (n) IP with anti-RAN followed by IB with anti-Exp-5, RCC1 and RAN antibodies in lysates of K562 and CML CD34 + . Comparison between groups was performed by two-tailed, unpaired Student’s t -test. P values ≤0.05 were considered significant. Results shown represent mean ± SEM. * p ≤ 0.05, ** p
    Cleaved Parp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 7715 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti parp
    (A) Resveratrol, rapamycin and a combination of the two inhibit mTORC1 and <t>mTORC2</t> signaling in the MM1.S cell line, as determined by immunoblot analysis. (B) Immunoblot analysis for caspase-3, <t>PARP,</t> p-Rb and cyclin D1 in MM1.S cells treated with resveratrol, rapamycin or a combination of the two. mTORC, mammalian transcriptional coactivator for CREB; PARP, poly (ADP-ribose) polymerase; p-Rb, phosphorylated retinoblastoma protein; Akt, protein kinase B.
    Anti Parp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 4480 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology parp1
    <t>PARP1</t> binds specifically to the FGF4 enhancer in vitro and in vivo . A , purification of FGF4 enhancer-binding proteins by affinity chromatography using biotin-labeled oligonucleotide of the FGF4 enhancer containing Oct4 and Sox2 binding sequence (biotin-
    Parp1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1235 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc parp 1
    RNF146 overexpression is cytoprotective in transiently transfected cells. (A, B) 293T cells were transiently transfected with CMV-RNF146 vector (CMV-RNF) or empty pcDNA3.1 + vector (CTL) and exposed to H 2 O 2 to induce <t>PARP-1</t> activation and cell death. (A) Cell viability was measured after 3 h by the MTT assay. (B) PARP-1 activation is measured at its the peak (5 min in 293T cells) by immunoblotting for poly(ADP-ribose) (PAR) polymer at 116 kDa. The PARP inhibitor PJ34 (3 μmol/L) was used in pretreatment (30 min). Representative blot image and densitometric analysis results are shown. (C) H9c2 cells were transiently transfected with pcDNA-RNF146 and exposed to H 2 O 2 (600 μmol/L, 30 min, peak of PARP activation in H9c2 cells). Cells were fixed and immunostained for RNF146 (left panel) and PAR polymer (right panel). Decreased PAR signal was detectable (right panel, arrow) in cells showing RNF146 overexpression (left panel, arrow).
    Parp 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1353 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti cleaved parp
    RNF146 overexpression is cytoprotective in transiently transfected cells. (A, B) 293T cells were transiently transfected with CMV-RNF146 vector (CMV-RNF) or empty pcDNA3.1 + vector (CTL) and exposed to H 2 O 2 to induce <t>PARP-1</t> activation and cell death. (A) Cell viability was measured after 3 h by the MTT assay. (B) PARP-1 activation is measured at its the peak (5 min in 293T cells) by immunoblotting for poly(ADP-ribose) (PAR) polymer at 116 kDa. The PARP inhibitor PJ34 (3 μmol/L) was used in pretreatment (30 min). Representative blot image and densitometric analysis results are shown. (C) H9c2 cells were transiently transfected with pcDNA-RNF146 and exposed to H 2 O 2 (600 μmol/L, 30 min, peak of PARP activation in H9c2 cells). Cells were fixed and immunostained for RNF146 (left panel) and PAR polymer (right panel). Decreased PAR signal was detectable (right panel, arrow) in cells showing RNF146 overexpression (left panel, arrow).
    Anti Cleaved Parp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1727 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson parp1
    Biochemical and cellular characteristics of natural <t>PARP1</t> variants. ( A ) An inherited PARP1 mutation identified by exome sequencing in a patient with pediatric colorectal cancer. The panel on the left shows a subset of the sequencing reads spanning the individual mutations (data based on hg19); the panel on the right shows the validation by Sanger sequencing in the child and the maternal samples to indicate the mode of inheritance. Position of the mutation is indicated by red arrows. ( B ) Biochemical characterization of natural PARP1 variants as used in this study. Rec. enzymes were expressed in the Sf 9/baculovirus system and purified via size exclusion and affinity chromatography. PARP1 activity was examined by incubating 5 nM PARP1 with increasing concentrations of NAD + as indicated in a reaction mixture as described in material and methods section. Afterward, 15% of reaction mixtures were slot-blotted on a nylon membrane (see Supplementary Figure S7) and PAR content was analyzed by immunochemical staining using the 10H antibody. Means of n = 3 independent experiments. A non-linear Michaelis-Menten model was used for curve fit. Statistical analysis using 2-way ANOVA testing. ( C ) Analysis of intracellular PARP1 activity in PARP1 KO cells reconstituted with PARP1 variants as indicated 2 days after transfection by immuno-epifluorescence microscopy as shown in Figure 4 (for representative raw data refer to Supplementary Figure S7). Cells were treated with H 2 O 2 for 5 min in concentrations as indicated, and PAR levels of eGFP-positive cells were examined using the anti-PAR-specific mAB 10H. Means ± SEM of n = 5 independent experiments. Statistical analysis was performed using matched two-way ANOVA testing and Sidak's post-test. ( D ) Time-course analysis of PAR formation in PARP1-reconstituted cells after treatment of cells with 250 μM H 2 O 2 . Means ± SEM of n = 4 independent experiments ( > 100 cells per experiment). Statistical analysis using matched two-way ANOVA testing and Sidak's post-test. ( E ) NAD + levels in WT, PARP1 KO and PARP1-reconstituted cells ± H 2 O 2 treatment for 7 min as evaluated by an enzymatic NAD + cycling assay. Means ± SEM of n = 3 independent experiments. Statistical analysis was performed via 2-way ANOVA testing and Sidak's post-test. ( F ) Recruitment and dissociation kinetics of natural PARP1 variants at sites of laser-induced DNA damage. For representative raw data refer to Supplementary Figure S7. Means ± SEM. Evaluation from ≥35 cells from three independent experiments. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test.
    Parp1, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology anti parp
    JNK2, but not JNK1, is essential for virus-induced apoptosis. ( A ) Control, JNK1 and JNK2 siRNA knock-down HEK293 cells ( left ), or wild type, Jnk1 −/− and Jnk2 −/− MEF cells ( right ), were treated with SeV (for HEK293 MOI = 1, for MEF MOI = 4) for the indicated times. Cell lysates were analyzed by western blot, probing for ISG15, ISG60, JNK1 and JNK2 with the indicated antibodies. ( B ) Wild type and Mavs −/− MEF cells were treated with SeV (MOI = 4), or TNF-α (10 ng/ml) plus cycloheximide (CHX, 10 µg/ml) for the indicated times. Cell lysates were collected for western blot analysis using <t>anti-PARP</t> antibody to determine cell apoptosis and using anti-MAVS antibody to measure the deficiency of MAVS protein. ( C ) HEK293 cells were transfected with the indicated plasmids and 24 hours later, cell lysates were collected for western blot analysis of PARP, phosphorylated JNK, phosphorylated IRF3, Flag-tagged proteins and β-actin. ( D ) HEK293 cells were treated by SeV (MOI = 1) with or without JNK kinase inhibitor SP600125 (5 µM). Cell lysates were collected for western blot analysis of PARP, cleaved <t>caspase-3</t> and β-actin to probe for cell apoptosis. ( E ) Control, JNK1 or JNK2 knocked down HEK293 cells were treated with SeV (MOI = 1) for the indicated times. Cell lysates were collected for western blot analysis to measure cell apoptosis using the indicated antibodies. ( F ) Wild type, Jnk1 −/− or Jnk2 −/− MEF cells were treated with SeV (MOI = 4) for the indicated times. Cell lysates were collected for western blot analysis.
    Anti Parp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 832 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Trevigen parp 1
    Effect of poly(ADP-ribosyl)ated <t>PARP-1</t> on DNA joining by DNA ligase III. (A) Purified PARP-1 (100 nM) was preincubated with a labeled DNA duplex containing a single ligatable nick (50 nM) in the presence (+) or absence (−) of NAD. Subsequently, intact DNA ligase III (Lig III; 5 nM) or a truncated version lacking the zinc finger (ΔZf-Lig III; 5 nM) was added to the reaction mixture as indicated. After incubation for 10 min at 25°C, labeled oligonucleotides were separated by denaturing gel electrophoresis. The labeled substrate (30-mer) and ligated product (50-mer) were detected and quantitated by phosphorimager analysis. (B) The results of three independent DNA joining assays are shown graphically. White bars, full-length DNA ligase IIIβ; grey bars, truncated version of DNA ligase IIIβ lacking the zinc finger.
    Parp 1, supplied by Trevigen, used in various techniques. Bioz Stars score: 92/100, based on 473 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam cleaved parp
    Fatostatin inhibits growth of and induces EnRS in MCF-7 xenograft tumors. MCF-7 cell xenograft tumors were initiated in athymic mice supplemented with estradiol capsules. Once tumors reached ~25 mm 2 , FS or DMSO control were administered daily ( n = 12–14 tumors/group). a Tumor size was measured and plotted over time. b Tumors were excised after 16 days of treatment and weighed. Animal body weight on day 16 is indicated. c–e Ki67 and cleaved <t>PARP</t> were examined in FFPE tumor sections by IHC and quantified. Bars represent 100 µm. f p-eIF2α was examined by IF with DAPI as a nuclear stain in DMSO and FS-treated tumors. g Quantitation of immunofluorescence was performed using the cell seed/spot segmentation analysis in ImageJ FIJI. The number of cells with color intensity +5% over background were counted as positive staining for p-eIF2α. h The mean color intensity of each cell staining positive for p-eIF2α was determined and plotted as number of cells vs. intensity. i <t>SREBP1</t> was examined by IF with DAPI as a nuclear stain in DMSO and FS-treated tumors. j The number of nuclei with SREBP1 staining was determined and plotted per 100 cells. * P
    Cleaved Parp, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 726 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson anti parp
    Targeting of <t>RAD51</t> activates several signaling pathways but attenuated by combining with <t>PARP</t> and p38 inhibition A. The Human Phospho-Kinase arrays (R D Systems) were probed with MDA-MB-231 lysate samples that had been treated for 72 hours; samples used are labeled (from top to bottom): (1) DMSO treatment, (2) 10 μM RAD51i, (3) 2.5 μM PARPi/10 μM p38i, and (4) 10 μM RAD51i /2.5 μM PARPi/10 μM p38i triple combination. Highlighted dots represent a significant change in signal over DMSO treated controls. The corners are positive control blots for quantification. B. Quantitation of spot intensity was standardized for cells treated with DMSO and plotted as normalized intensity. Several kinases displayed greater than 2 fold increase in phosphorylation compared to references. Shading represents grouping based on pathway signaling. C. Protein expression and changes in phosphorylation of ERK1/2, p38, STAT3, MK-2 (p38 target) and AKT were confirmed by western blotting.
    Anti Parp, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 412 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    The Jackson Laboratory parp 1
    Schematic illustration of the impact of <t>PARP-1</t> loss of function leading to EMT induction and aggressive prostate tumor growth, potentially mediated by Smad-directed TGF-β signaling. A potential cross-effect by nuclear AR depletion may contribute to enhanced EMT.
    Parp 1, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 94/100, based on 727 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    parp1  (Abcam)
    99
    Abcam parp1
    Mechanism of tumor suppressive function of CLDN7 in ccRCC. ( A ) Heatmap representation of differentially expressed genes identified by RNA-Seq between Caki-1 CLDN7 cells ( n = 3) and Caki-1 Control cells (n = 3). ( B ) Statistics of KEGG pathway enrichment. The y-axis corresponds to KEGG Pathway, and the x-axis shows the GeneRatio. The color of the dot represent adjusted p value (padj), and the size of the dot represents the number of differentially expressed genes mapped to the reference pathways. ( C ) Validation of differentially expressed genes by qRT-PCR. Comparison of mRNA expression of genes in pathways of cancer ( BCL2 , HIF1A , GLI-1 , ITGB-1 , p21 and AR ) and genes in EMT-related pathway ( TGFB1 , E-cadherin , N-cadherin , Vimentin and TWIST1 ) between Caki-1 CLDN7 cells and Caki-1 Control cells. All data are shown as means ± SD. ( D ) a . Western blot assay (left) and statistical analysis (right) of CLDN7, BCL2, <t>cleaved-PARP1</t> and cleaved-Caspase 3 expression in Caki-1 and A498 cells, while overexpression of CLDN7 compared with control group. b . IHC assay of BCL2, cleaved-Caspase 3, Ki-67 and CLDN7 expression in xenografts formed by Caki-1 CLDN7 and Control cells. Scale bar, 100 μm. ( E ) a . Western blot assay and statistical analysis of E-cadherin, N-cadherin and Vimentin expression in CLDN7 overexpressed Caki-1 and A498 cells, comparing with control cells. b . IHC assay of E-cadherin, N-cadherin, TGFB1 and CLDN7 in xenografts formed by Caki-1 CLDN7 and Control cells. Scale bar, 100 μm. N.S, not significant. *p
    Parp1, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 92 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology cleaved parp
    The effect of basic fibroblast growth factor (bFGF) on mitochondrial dysfunction-related proteins induced by hydroperoxide (TBHP) in H9C2 cells. (A) H9C2 cells were pre-treated with 50 ng/ml bFGF for 2 hrs, and then 100 μM TBHP was added for an additional 8 hrs. The cell lysates were analysed for the expression of Bax, Bcl-2, <t>cleaved-PARP</t> and <t>cleaved-caspase-9</t> by western blotting. Bar diagram of Bax, Bcl-2, cleaved-PARP and cleaved-caspase-9 expression from three Western blot analyses. (B) Immunofluorescence results of the mitochondrial apoptotic marker cytochrome c in H9C2 cells. * P
    Cleaved Parp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 491 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology anti parp 1
    3-AB inhibits mouse MOR mRNA expression in NS20Y cells and schematic model for <t>PARP-1</t> in modulation of mouse MOR transcription. (A) Quantification of transcripts was performed by RT-PCR. Total RNA from NS20Y cells treated with 2 mM 3-AB was prepared and treated with DNase I. Primer pairs specific for the coding sequence of each gene were used for RT-PCR. PCR products were visualized in a 2% agarose gel. Lane 1: Molecular weight markers (M); lane 2: Control; lane 3: 3-AB-treated cells. (B) Quantitative analysis using ImageQuant 5.2 software. The MOR mRNA levels from Control and 3-AB-treated cells were normalized against β-actin levels. The values were obtained from triplicate data points. Changes in transcript levels for 3-AB-treated samples were compared to Control, which was assigned a value of 1.0. Bars indicate the range of standard error. (C) Schematic model for the role of PARP-1 in modulation of mouse MOR gene transcription. In neuronal cells, enzymatically active PARP-1 interacts strongly with the poly(C) sequence of the mouse MOR promoter and aids in the formation of tran-scriptionally inactive chromatin. Enzymatic inhibition of PARP-1 by 3-AB results in non-poly(ADP-ribosyl)ated PARP-1 and subsequently, an increase in the levels of MOR mRNA in mouse NS20Y cells.
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    99
    Cell Signaling Technology Inc cleaved poly
    3-AB inhibits mouse MOR mRNA expression in NS20Y cells and schematic model for <t>PARP-1</t> in modulation of mouse MOR transcription. (A) Quantification of transcripts was performed by RT-PCR. Total RNA from NS20Y cells treated with 2 mM 3-AB was prepared and treated with DNase I. Primer pairs specific for the coding sequence of each gene were used for RT-PCR. PCR products were visualized in a 2% agarose gel. Lane 1: Molecular weight markers (M); lane 2: Control; lane 3: 3-AB-treated cells. (B) Quantitative analysis using ImageQuant 5.2 software. The MOR mRNA levels from Control and 3-AB-treated cells were normalized against β-actin levels. The values were obtained from triplicate data points. Changes in transcript levels for 3-AB-treated samples were compared to Control, which was assigned a value of 1.0. Bars indicate the range of standard error. (C) Schematic model for the role of PARP-1 in modulation of mouse MOR gene transcription. In neuronal cells, enzymatically active PARP-1 interacts strongly with the poly(C) sequence of the mouse MOR promoter and aids in the formation of tran-scriptionally inactive chromatin. Enzymatic inhibition of PARP-1 by 3-AB results in non-poly(ADP-ribosyl)ated PARP-1 and subsequently, an increase in the levels of MOR mRNA in mouse NS20Y cells.
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    Image Search Results


    BCR-ABL deregulates miR-126 biogenesis (a,b) pri-miR-126 (n=8 biologically independent samples) (a) and pre-miR-126 (n=8 biologically independent samples) (b) expression levels, as assessed by QPCR, in the indicated human normal and CML cell populations. (c) BCR-ABL and SPRED1 staining in CML CD34 + cells by immunofluorescence (IF). (d) Immunoprecipitation (IP) with anti-SPRED1 followed by immunoblotting (IB) with anti-SPRED1 and anti-phosphotyrosine (p-Tyr) antibodies (left) and an in vitro kinase assay (right), as performed by IP with anti-c-Abl or anti-normal mouse IgG as control and immunoblotting with anti-SPRED1, in lysates of K562 cells treated with none, DMSO (vehicle) or NIL. (e) IP with anti-RAN followed by IB with anti-SPRED1 and anti-RAN antibodies in lysates of K562 cells. (f) SPRED1 and RAN staining by IF in K562 cells treated with none, DMSO or NIL. (g) SPRED1, RAN, RCC1 and Exp-5 expression in cytoplasmic (Cyt) and nuclear (Nu) fractions from K562 cells, treated with DMSO or NIL, as assessed by IB. Densitometric quantification of selected bands is shown (normalized to the actin loading control for total and Cyt lysates or to the PARP loading control for Nu lysates). (h) IP with anti-RAN followed by IB with anti-SPRED1, RAN, Exp-5 and RCC1 antibodies in lysates of K562 cells, CML CD34 + cells, and normal CD34+ cells treated with DMSO or NIL. Densitometric quantification of selected bands is shown (normalized to the actin loading control). (i) Mature, pri- and pre-miR-126 expression, as assessed by QPCR, in K562 and CML CD34 + cells treated with DMSO or NIL (n=3 independent experiments for K562 and 3 independent samples for CML cells). (j) IP with anti-RAN followed by IB with anti-SPRED1, Exp-5, RCC1 and RAN antibodies in lysates of K562 cells without or with washing-off of NIL. Densitometric quantification of selected bands is shown (normalized to the actin loading control). (k–m) Mature and pri-miR-126 expression as assessed by QPCR (n=3 independent experiments) (k), miR-126 staining (l), and mature and pre-miR-126 levels as assessed by Northern blotting (m) in K562 cells with or without washing-off of NIL. (n) IP with anti-RAN followed by IB with anti-Exp-5, RCC1 and RAN antibodies in lysates of K562 and CML CD34 + . Comparison between groups was performed by two-tailed, unpaired Student’s t -test. P values ≤0.05 were considered significant. Results shown represent mean ± SEM. * p ≤ 0.05, ** p

    Journal: Nature medicine

    Article Title: Bone Marrow Niche Trafficking of miR-126 Controls Self-Renewal of Leukemia Stem Cells in Chronic Myelogenous Leukemia

    doi: 10.1038/nm.4499

    Figure Lengend Snippet: BCR-ABL deregulates miR-126 biogenesis (a,b) pri-miR-126 (n=8 biologically independent samples) (a) and pre-miR-126 (n=8 biologically independent samples) (b) expression levels, as assessed by QPCR, in the indicated human normal and CML cell populations. (c) BCR-ABL and SPRED1 staining in CML CD34 + cells by immunofluorescence (IF). (d) Immunoprecipitation (IP) with anti-SPRED1 followed by immunoblotting (IB) with anti-SPRED1 and anti-phosphotyrosine (p-Tyr) antibodies (left) and an in vitro kinase assay (right), as performed by IP with anti-c-Abl or anti-normal mouse IgG as control and immunoblotting with anti-SPRED1, in lysates of K562 cells treated with none, DMSO (vehicle) or NIL. (e) IP with anti-RAN followed by IB with anti-SPRED1 and anti-RAN antibodies in lysates of K562 cells. (f) SPRED1 and RAN staining by IF in K562 cells treated with none, DMSO or NIL. (g) SPRED1, RAN, RCC1 and Exp-5 expression in cytoplasmic (Cyt) and nuclear (Nu) fractions from K562 cells, treated with DMSO or NIL, as assessed by IB. Densitometric quantification of selected bands is shown (normalized to the actin loading control for total and Cyt lysates or to the PARP loading control for Nu lysates). (h) IP with anti-RAN followed by IB with anti-SPRED1, RAN, Exp-5 and RCC1 antibodies in lysates of K562 cells, CML CD34 + cells, and normal CD34+ cells treated with DMSO or NIL. Densitometric quantification of selected bands is shown (normalized to the actin loading control). (i) Mature, pri- and pre-miR-126 expression, as assessed by QPCR, in K562 and CML CD34 + cells treated with DMSO or NIL (n=3 independent experiments for K562 and 3 independent samples for CML cells). (j) IP with anti-RAN followed by IB with anti-SPRED1, Exp-5, RCC1 and RAN antibodies in lysates of K562 cells without or with washing-off of NIL. Densitometric quantification of selected bands is shown (normalized to the actin loading control). (k–m) Mature and pri-miR-126 expression as assessed by QPCR (n=3 independent experiments) (k), miR-126 staining (l), and mature and pre-miR-126 levels as assessed by Northern blotting (m) in K562 cells with or without washing-off of NIL. (n) IP with anti-RAN followed by IB with anti-Exp-5, RCC1 and RAN antibodies in lysates of K562 and CML CD34 + . Comparison between groups was performed by two-tailed, unpaired Student’s t -test. P values ≤0.05 were considered significant. Results shown represent mean ± SEM. * p ≤ 0.05, ** p

    Article Snippet: Primary antibodies included anti-SPRED1 antibody (M23-P2G3, #ab64740, Abcam), anti-RAN antibody (C-20, #SC-1156, Santa Cruz), anti-Exportin5 antibody (D7W6W, #12565, Cell Signaling), anti-PARP antibody (#9542, Cell Signaling), anti-RCC1 antibody (F-2, #SC-376049, Santa Cruz), anti-Actin antibody (C-4, #SC-47778, Santa Cruz), anti-phospho-Tyrosine antibody (4G10, #05-321, Millipore), anti-BCL-2 antibody (124, #15071, Cell Signaling), anti-phospho-ERK (#9101, Cell Signaling), anti-normal mouse IgG (#SC-2025, Santa Cruz), anti-normal rabbit IgG (#SC-2027, Santa Cruz), CD63 (10628D, ThermoFisher), TSG101(SAB2702167, Sigma), HSP90 (2D12, Enzo Life Sciences), Cytochrome C (sc-13156, Santa Cruz).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Staining, Immunofluorescence, Immunoprecipitation, In Vitro, Kinase Assay, Northern Blot, Two Tailed Test

    (A) Resveratrol, rapamycin and a combination of the two inhibit mTORC1 and mTORC2 signaling in the MM1.S cell line, as determined by immunoblot analysis. (B) Immunoblot analysis for caspase-3, PARP, p-Rb and cyclin D1 in MM1.S cells treated with resveratrol, rapamycin or a combination of the two. mTORC, mammalian transcriptional coactivator for CREB; PARP, poly (ADP-ribose) polymerase; p-Rb, phosphorylated retinoblastoma protein; Akt, protein kinase B.

    Journal: Oncology Letters

    Article Title: Combining the mammalian target of rapamycin inhibitor, rapamycin, with resveratrol has a synergistic effect in multiple myeloma

    doi: 10.3892/ol.2018.8178

    Figure Lengend Snippet: (A) Resveratrol, rapamycin and a combination of the two inhibit mTORC1 and mTORC2 signaling in the MM1.S cell line, as determined by immunoblot analysis. (B) Immunoblot analysis for caspase-3, PARP, p-Rb and cyclin D1 in MM1.S cells treated with resveratrol, rapamycin or a combination of the two. mTORC, mammalian transcriptional coactivator for CREB; PARP, poly (ADP-ribose) polymerase; p-Rb, phosphorylated retinoblastoma protein; Akt, protein kinase B.

    Article Snippet: Polyvinylidene difluoride membranes were incubated in 5% non-fat dry milk at room temperature for 2 h. Rabbit anti-mTORC1 (cat. no. 2587; 1:1,000), mouse anti-mTORC2 (cat. no. 13017; 1:1,000), rabbit anti-caspase-3 (cat. no. 9664; 1:1,000) and rabbit anti-PARP (cat. no. 9532; 1:1,000) antibodies (Cell Signaling Technology, Inc.) were added and incubated overnight at 4°C.

    Techniques:

    H1.2 PARylation permits its displacement from chromatin upon DNA damage. a HeLa cells were transfected with GFP-H1.2 and treated with 20 μM Ku55933 or 2 μM Ku57788 for 4 h or 5 μM PJ34 for 1 h followed by laser micro-irradiation. Images were taken every 10 s for 5 min and quantifications of the IR path signal intensity were shown and ~15 IR paths from 10 separate cells were calculated. The data represent the mean ± SD. Scale bars, 10 μm. b HeLa cells were transfected with the indicated siRNAs and treated with 40 μM etoposide for the indicated time. Chromatin was fractionated and analyzed by immunoblotting. c Parp1 wild-type (+/+) or KO (−/−) MEFs were treated with 40 μM etoposide for the indicated time and chromatin was fractionated and analyzed by immunoblotting. d HeLa cells were transfected with FLAG-H1.2 and treated with 40 μM etoposide for 15 min with or without 5 μM PJ34 for 1 h. Cell extracts were immunoprecipitated with FLAG-conjugated M2 beads. e Recombinant HIS-H1.2 was subjected to in vitro PARylation assay in the presence of NAD + or 10 μM PJ34, as indicated. f HeLa cells were transfected with wild-type or S188A mutated FLAG-H1.2 and treated with 40 μM etoposide for 15 min with or without 5 μM PJ34 for 1 h, as indicated. Cells were extracted and immunoprecipitated with FLAG-conjugated M2 beads. g Recombinant wild-type, S188A mutated or C1-deleted (ΔC1) HIS-H1.2 were subjected to in vitro PARylation assay. h HeLa cells were transfected with wild-type, ΔC1 or S188A mutated GFP-H1.2 and subjected to laser micro-irradiation. Images were taken every 20 s for 5 min and representative images were shown. Quantifications were calculated as in a . The data represent the mean ± SD. Scale bars, 10 μm

    Journal: Cell Research

    Article Title: Destabilization of linker histone H1.2 is essential for ATM activation and DNA damage repair

    doi: 10.1038/s41422-018-0048-0

    Figure Lengend Snippet: H1.2 PARylation permits its displacement from chromatin upon DNA damage. a HeLa cells were transfected with GFP-H1.2 and treated with 20 μM Ku55933 or 2 μM Ku57788 for 4 h or 5 μM PJ34 for 1 h followed by laser micro-irradiation. Images were taken every 10 s for 5 min and quantifications of the IR path signal intensity were shown and ~15 IR paths from 10 separate cells were calculated. The data represent the mean ± SD. Scale bars, 10 μm. b HeLa cells were transfected with the indicated siRNAs and treated with 40 μM etoposide for the indicated time. Chromatin was fractionated and analyzed by immunoblotting. c Parp1 wild-type (+/+) or KO (−/−) MEFs were treated with 40 μM etoposide for the indicated time and chromatin was fractionated and analyzed by immunoblotting. d HeLa cells were transfected with FLAG-H1.2 and treated with 40 μM etoposide for 15 min with or without 5 μM PJ34 for 1 h. Cell extracts were immunoprecipitated with FLAG-conjugated M2 beads. e Recombinant HIS-H1.2 was subjected to in vitro PARylation assay in the presence of NAD + or 10 μM PJ34, as indicated. f HeLa cells were transfected with wild-type or S188A mutated FLAG-H1.2 and treated with 40 μM etoposide for 15 min with or without 5 μM PJ34 for 1 h, as indicated. Cells were extracted and immunoprecipitated with FLAG-conjugated M2 beads. g Recombinant wild-type, S188A mutated or C1-deleted (ΔC1) HIS-H1.2 were subjected to in vitro PARylation assay. h HeLa cells were transfected with wild-type, ΔC1 or S188A mutated GFP-H1.2 and subjected to laser micro-irradiation. Images were taken every 20 s for 5 min and representative images were shown. Quantifications were calculated as in a . The data represent the mean ± SD. Scale bars, 10 μm

    Article Snippet: The antibodies used in this study include: anti-ATM (GeneTex, GTX70103), anti-H1.2 (GeneTex, GTX122561), anti-GFP (MBL, M048-3), anti-HIS (MBL, PM032), anti-HA (MBL, M180-3), anti-FLAG (Sigma-Aldrich, F3165), anti-GST (APPLYGEN, C1303), anti-phospho-ATM (S1981; Cell Signaling, 5883), anti-phospho-NBS1 (S343; Cell Signaling, 3001), anti-phospho-SMC1 (S957; Cell Signaling, 58052), anti-SMC1 (Cell Signaling, 4802), anti-phospho-CHK2 (T68; Cell Signaling, 2197), anti-CHK2 (Cell Signaling, 3440), anti-phospho-p53 (S15; Cell Signaling, 9286), anti-phospho-H2AX (S139; Cell Signaling, 9718), anti-H2AX (Cell Signaling, 2595), anti-PARP1 (Cell Signaling, 9532), anti-GAPDH (Santa Cruz, sc-32233), anti-ATR (Santa Cruz, sc-1887), anti-DNA-PKcs (Santa Cruz, sc-1552), anti-NBS1 (Santa Cruz, sc-8580), anti-p53 (Santa Cruz, sc-126), anti-H1.4 (Santa Cruz, sc-34464), anti-cyclin E (Santa Cruz, sc-247), anti-H1.2 (for IP and ChIP; Abcam, ab17677), anti-H1.3 (Abcam, ab24174), anti-H3 (Abcam, ab1791), anti-H4 (Abcam, ab10158), anti-RAD50 (Abcam, ab89), anti-MRE11 (Abcam, ab12159), anti-phospho-H3 (S10; Abcam, ab5176), anti-ace-H3 (Active Motif, 39139), anti-H1 (Active Motif, 39707), anti-PAR (Trevigen, 4335-MC-100).

    Techniques: Transfection, Irradiation, Immunoprecipitation, Recombinant, In Vitro

    PARylation of H1.2 is essential for ATM activation. a Parp1 wild-type (+/+) or KO (−/−) MEFs were treated with 40 μM etoposide for the indicated time and analyzed by immunoblotting. b HeLa cells were treated with 40 μM etoposide for the indicated time with or without exposure to 5 μM PJ34 1 h before etoposide treatment and analyzed by immunoblotting. c Two clones of PARP1 stable knockdown (shPARP1 #1 and #3) and control (shCtr) HeLa cells were treated with 40 μM etoposide for 30 min and analyzed by immunoblotting. d shPARP1 (1#) and shCtr HeLa cells were transfected with the indicated siRNAs and treated with 40 μM etoposide for 30 min and analyzed by immunoblotting. e HCT116 cells were transfected with the indicated plasmids and treated with 40 μM etoposide for the indicated times and analyzed by immunoblotting. f HeLa cells were transfected with wild-type or S188A mutated GFP-H1.2, treated with 40 μM etoposide for 2 h and the fluorescence intensity of phospho-ATM S1981 in the untransfected cells was normalized to 1. The arrows indicate representative cells. The data represent the mean ± SD. Scale bars, 10 μm. g Recombinant HIS-H1.2 was incubated for 30 min at 37 °C with PARP1 with or without NAD + for in vitro PARylation assay (Incubation 1, Inc. 1). H1.2 was eluted and used for in vitro phosphorylation assay (Incubation 2, Inc. 2). An N-terminal GST-p53 (1–99 aa) peptide was used as the substrate. h Recombinant GST-H1.2 was incubated with PARP1 with or without NAD + for in vitro PARylation assay. GST alone and PARylated GST-H1.2 were then incubated with HIS-MRE11 for GST-pulldown assay. * indicates specific protein bands. i HeLa cells were transfected with the indicated plasmids and treated with 40 μM etoposide for 1 h or 5 μM PJ34 for 1 h. Whole cell extractions were prepared and subjected to Co-IP assay with FLAG-conjugated M2 beads

    Journal: Cell Research

    Article Title: Destabilization of linker histone H1.2 is essential for ATM activation and DNA damage repair

    doi: 10.1038/s41422-018-0048-0

    Figure Lengend Snippet: PARylation of H1.2 is essential for ATM activation. a Parp1 wild-type (+/+) or KO (−/−) MEFs were treated with 40 μM etoposide for the indicated time and analyzed by immunoblotting. b HeLa cells were treated with 40 μM etoposide for the indicated time with or without exposure to 5 μM PJ34 1 h before etoposide treatment and analyzed by immunoblotting. c Two clones of PARP1 stable knockdown (shPARP1 #1 and #3) and control (shCtr) HeLa cells were treated with 40 μM etoposide for 30 min and analyzed by immunoblotting. d shPARP1 (1#) and shCtr HeLa cells were transfected with the indicated siRNAs and treated with 40 μM etoposide for 30 min and analyzed by immunoblotting. e HCT116 cells were transfected with the indicated plasmids and treated with 40 μM etoposide for the indicated times and analyzed by immunoblotting. f HeLa cells were transfected with wild-type or S188A mutated GFP-H1.2, treated with 40 μM etoposide for 2 h and the fluorescence intensity of phospho-ATM S1981 in the untransfected cells was normalized to 1. The arrows indicate representative cells. The data represent the mean ± SD. Scale bars, 10 μm. g Recombinant HIS-H1.2 was incubated for 30 min at 37 °C with PARP1 with or without NAD + for in vitro PARylation assay (Incubation 1, Inc. 1). H1.2 was eluted and used for in vitro phosphorylation assay (Incubation 2, Inc. 2). An N-terminal GST-p53 (1–99 aa) peptide was used as the substrate. h Recombinant GST-H1.2 was incubated with PARP1 with or without NAD + for in vitro PARylation assay. GST alone and PARylated GST-H1.2 were then incubated with HIS-MRE11 for GST-pulldown assay. * indicates specific protein bands. i HeLa cells were transfected with the indicated plasmids and treated with 40 μM etoposide for 1 h or 5 μM PJ34 for 1 h. Whole cell extractions were prepared and subjected to Co-IP assay with FLAG-conjugated M2 beads

    Article Snippet: The antibodies used in this study include: anti-ATM (GeneTex, GTX70103), anti-H1.2 (GeneTex, GTX122561), anti-GFP (MBL, M048-3), anti-HIS (MBL, PM032), anti-HA (MBL, M180-3), anti-FLAG (Sigma-Aldrich, F3165), anti-GST (APPLYGEN, C1303), anti-phospho-ATM (S1981; Cell Signaling, 5883), anti-phospho-NBS1 (S343; Cell Signaling, 3001), anti-phospho-SMC1 (S957; Cell Signaling, 58052), anti-SMC1 (Cell Signaling, 4802), anti-phospho-CHK2 (T68; Cell Signaling, 2197), anti-CHK2 (Cell Signaling, 3440), anti-phospho-p53 (S15; Cell Signaling, 9286), anti-phospho-H2AX (S139; Cell Signaling, 9718), anti-H2AX (Cell Signaling, 2595), anti-PARP1 (Cell Signaling, 9532), anti-GAPDH (Santa Cruz, sc-32233), anti-ATR (Santa Cruz, sc-1887), anti-DNA-PKcs (Santa Cruz, sc-1552), anti-NBS1 (Santa Cruz, sc-8580), anti-p53 (Santa Cruz, sc-126), anti-H1.4 (Santa Cruz, sc-34464), anti-cyclin E (Santa Cruz, sc-247), anti-H1.2 (for IP and ChIP; Abcam, ab17677), anti-H1.3 (Abcam, ab24174), anti-H3 (Abcam, ab1791), anti-H4 (Abcam, ab10158), anti-RAD50 (Abcam, ab89), anti-MRE11 (Abcam, ab12159), anti-phospho-H3 (S10; Abcam, ab5176), anti-ace-H3 (Active Motif, 39139), anti-H1 (Active Motif, 39707), anti-PAR (Trevigen, 4335-MC-100).

    Techniques: Activation Assay, Clone Assay, Transfection, Fluorescence, Recombinant, Incubation, In Vitro, Phosphorylation Assay, GST Pulldown Assay, Co-Immunoprecipitation Assay

    Linker histone H1.2 dissociation and destabilization are required for DNA repair and cell survival. a , b Wild-type and H1.2 KO (1# and 2#) HeLa cells were analyzed by comet and colony formation assays. The tail moment of wild-type cells at 10 min post treatment was normalized to 1. The data represent the mean ± SD. c Wild-type and H1.2 KO (1# and 2#) DR-GFP U2OS cells were analyzed by DR-GFP assay. The data represent the mean ± SD. d Wild-type and H1.2 KO pEJ5-GFP U2OS cells were analyzed by EJ5-GFP assay. The data represent the mean ± SD. e , f Wild-type, H1.2 KO (1#), two ATM KO (1# and 3#) and two ATM/H1.2 double KO (5# and 6#) HeLa cells were analyzed by comet and colony formation assays. The tail moment of wild type cells at 12 h post treatment was normalized to 1. The data represent the mean ± SD. g , h Wild-type, H1.2 KO (1#), H1.2 KO (1#) with reintroduced wild type or S188A mutated H1.2, and ATM KO (1#) HeLa cells were analyzed by comet and colony formation assays. ATM KO (1#) HeLa cells with reintroduced wild-type or S188A mutated H1.2 were also analyzed by colony formation assay. The tail moment of wild-type cells at 10 min post treatment was normalized to 1. The data represent the mean ± SD. i A schematic model for the dynamic regulation of ATM by H1.2. In the absence of DNA damage, H1.2 binds to the chromatin and blocks the interactions between ATM and MRN to prevent the recruitment and activation of ATM. Upon DNA damage, PARP1 is activated to PARylate and displace H1.2 from chromatin, whereby ATM is permitted to be recruited and activated by MRN and DNA breaks. Activated ATM, which is amplified by an ATM-MDC1-MRN positive feedback loop, drives the DNA damage response through phosphorylation of a wide spectrum of substrates, including H2AX

    Journal: Cell Research

    Article Title: Destabilization of linker histone H1.2 is essential for ATM activation and DNA damage repair

    doi: 10.1038/s41422-018-0048-0

    Figure Lengend Snippet: Linker histone H1.2 dissociation and destabilization are required for DNA repair and cell survival. a , b Wild-type and H1.2 KO (1# and 2#) HeLa cells were analyzed by comet and colony formation assays. The tail moment of wild-type cells at 10 min post treatment was normalized to 1. The data represent the mean ± SD. c Wild-type and H1.2 KO (1# and 2#) DR-GFP U2OS cells were analyzed by DR-GFP assay. The data represent the mean ± SD. d Wild-type and H1.2 KO pEJ5-GFP U2OS cells were analyzed by EJ5-GFP assay. The data represent the mean ± SD. e , f Wild-type, H1.2 KO (1#), two ATM KO (1# and 3#) and two ATM/H1.2 double KO (5# and 6#) HeLa cells were analyzed by comet and colony formation assays. The tail moment of wild type cells at 12 h post treatment was normalized to 1. The data represent the mean ± SD. g , h Wild-type, H1.2 KO (1#), H1.2 KO (1#) with reintroduced wild type or S188A mutated H1.2, and ATM KO (1#) HeLa cells were analyzed by comet and colony formation assays. ATM KO (1#) HeLa cells with reintroduced wild-type or S188A mutated H1.2 were also analyzed by colony formation assay. The tail moment of wild-type cells at 10 min post treatment was normalized to 1. The data represent the mean ± SD. i A schematic model for the dynamic regulation of ATM by H1.2. In the absence of DNA damage, H1.2 binds to the chromatin and blocks the interactions between ATM and MRN to prevent the recruitment and activation of ATM. Upon DNA damage, PARP1 is activated to PARylate and displace H1.2 from chromatin, whereby ATM is permitted to be recruited and activated by MRN and DNA breaks. Activated ATM, which is amplified by an ATM-MDC1-MRN positive feedback loop, drives the DNA damage response through phosphorylation of a wide spectrum of substrates, including H2AX

    Article Snippet: The antibodies used in this study include: anti-ATM (GeneTex, GTX70103), anti-H1.2 (GeneTex, GTX122561), anti-GFP (MBL, M048-3), anti-HIS (MBL, PM032), anti-HA (MBL, M180-3), anti-FLAG (Sigma-Aldrich, F3165), anti-GST (APPLYGEN, C1303), anti-phospho-ATM (S1981; Cell Signaling, 5883), anti-phospho-NBS1 (S343; Cell Signaling, 3001), anti-phospho-SMC1 (S957; Cell Signaling, 58052), anti-SMC1 (Cell Signaling, 4802), anti-phospho-CHK2 (T68; Cell Signaling, 2197), anti-CHK2 (Cell Signaling, 3440), anti-phospho-p53 (S15; Cell Signaling, 9286), anti-phospho-H2AX (S139; Cell Signaling, 9718), anti-H2AX (Cell Signaling, 2595), anti-PARP1 (Cell Signaling, 9532), anti-GAPDH (Santa Cruz, sc-32233), anti-ATR (Santa Cruz, sc-1887), anti-DNA-PKcs (Santa Cruz, sc-1552), anti-NBS1 (Santa Cruz, sc-8580), anti-p53 (Santa Cruz, sc-126), anti-H1.4 (Santa Cruz, sc-34464), anti-cyclin E (Santa Cruz, sc-247), anti-H1.2 (for IP and ChIP; Abcam, ab17677), anti-H1.3 (Abcam, ab24174), anti-H3 (Abcam, ab1791), anti-H4 (Abcam, ab10158), anti-RAD50 (Abcam, ab89), anti-MRE11 (Abcam, ab12159), anti-phospho-H3 (S10; Abcam, ab5176), anti-ace-H3 (Active Motif, 39139), anti-H1 (Active Motif, 39707), anti-PAR (Trevigen, 4335-MC-100).

    Techniques: Colony Assay, Activation Assay, Amplification

    PARP1 binds specifically to the FGF4 enhancer in vitro and in vivo . A , purification of FGF4 enhancer-binding proteins by affinity chromatography using biotin-labeled oligonucleotide of the FGF4 enhancer containing Oct4 and Sox2 binding sequence (biotin-

    Journal: The Journal of Biological Chemistry

    Article Title: PARP1 Poly(ADP-ribosyl)ates Sox2 to Control Sox2 Protein Levels and FGF4 Expression during Embryonic Stem Cell Differentiation *

    doi: 10.1074/jbc.M109.033118

    Figure Lengend Snippet: PARP1 binds specifically to the FGF4 enhancer in vitro and in vivo . A , purification of FGF4 enhancer-binding proteins by affinity chromatography using biotin-labeled oligonucleotide of the FGF4 enhancer containing Oct4 and Sox2 binding sequence (biotin-

    Article Snippet: We also determined the expression of FGF4 in PARP1 +/+ and PARP1 −/− cells treated with PJ34 or 3AB at different concentrations, and we found that both inhibitors caused a decrease in the mRNA level of FGF4 in differentiating PARP1 +/+ cells at a higher dosage but caused less of a decrease in PARP1 −/− cells ( C and ).

    Techniques: In Vitro, In Vivo, Purification, Binding Assay, Affinity Chromatography, Labeling, Sequencing

    Sox2 is poly(ADP-ribosyl)ated in vivo and in vitro . A , poly(ADP-ribosyl)ation levels of PARP1 +/+ and PARP1 − / − cell extracts were evaluated using anti-PAR antibody. PARP1 +/+ and PARP1 − / − ESCs were cultured without a feeder

    Journal: The Journal of Biological Chemistry

    Article Title: PARP1 Poly(ADP-ribosyl)ates Sox2 to Control Sox2 Protein Levels and FGF4 Expression during Embryonic Stem Cell Differentiation *

    doi: 10.1074/jbc.M109.033118

    Figure Lengend Snippet: Sox2 is poly(ADP-ribosyl)ated in vivo and in vitro . A , poly(ADP-ribosyl)ation levels of PARP1 +/+ and PARP1 − / − cell extracts were evaluated using anti-PAR antibody. PARP1 +/+ and PARP1 − / − ESCs were cultured without a feeder

    Article Snippet: We also determined the expression of FGF4 in PARP1 +/+ and PARP1 −/− cells treated with PJ34 or 3AB at different concentrations, and we found that both inhibitors caused a decrease in the mRNA level of FGF4 in differentiating PARP1 +/+ cells at a higher dosage but caused less of a decrease in PARP1 −/− cells ( C and ).

    Techniques: In Vivo, In Vitro, Cell Culture

    PARP1 modulates the Sox2 protein level to control FGF4 expression. A , Sox2 protein level is markedly elevated in differentiating PARP1 − / − ESCs. ESCs were cultured under the differentiation condition for different lengths of time, as indicated,

    Journal: The Journal of Biological Chemistry

    Article Title: PARP1 Poly(ADP-ribosyl)ates Sox2 to Control Sox2 Protein Levels and FGF4 Expression during Embryonic Stem Cell Differentiation *

    doi: 10.1074/jbc.M109.033118

    Figure Lengend Snippet: PARP1 modulates the Sox2 protein level to control FGF4 expression. A , Sox2 protein level is markedly elevated in differentiating PARP1 − / − ESCs. ESCs were cultured under the differentiation condition for different lengths of time, as indicated,

    Article Snippet: We also determined the expression of FGF4 in PARP1 +/+ and PARP1 −/− cells treated with PJ34 or 3AB at different concentrations, and we found that both inhibitors caused a decrease in the mRNA level of FGF4 in differentiating PARP1 +/+ cells at a higher dosage but caused less of a decrease in PARP1 −/− cells ( C and ).

    Techniques: Expressing, Cell Culture

    PARP1 is implicated in FGF4 expression. A and B , qPCR analysis of gene expression in undifferentiated and differentiating PARP1 +/+ and PARP1 − / − ESCs cultured without a feeder layer and treated with 1 μ m RA. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: PARP1 Poly(ADP-ribosyl)ates Sox2 to Control Sox2 Protein Levels and FGF4 Expression during Embryonic Stem Cell Differentiation *

    doi: 10.1074/jbc.M109.033118

    Figure Lengend Snippet: PARP1 is implicated in FGF4 expression. A and B , qPCR analysis of gene expression in undifferentiated and differentiating PARP1 +/+ and PARP1 − / − ESCs cultured without a feeder layer and treated with 1 μ m RA. *, p

    Article Snippet: We also determined the expression of FGF4 in PARP1 +/+ and PARP1 −/− cells treated with PJ34 or 3AB at different concentrations, and we found that both inhibitors caused a decrease in the mRNA level of FGF4 in differentiating PARP1 +/+ cells at a higher dosage but caused less of a decrease in PARP1 −/− cells ( C and ).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Cell Culture

    A putative mechanism for PARP1 in control of FGF4 expression. A , ChIP analysis of association of Sox2 with the FGF4 or Sox2 enhancer in PARP1 +/+ and PARP1 − / − ESCs cultured under the differentiation condition. B , association of Sox2 with

    Journal: The Journal of Biological Chemistry

    Article Title: PARP1 Poly(ADP-ribosyl)ates Sox2 to Control Sox2 Protein Levels and FGF4 Expression during Embryonic Stem Cell Differentiation *

    doi: 10.1074/jbc.M109.033118

    Figure Lengend Snippet: A putative mechanism for PARP1 in control of FGF4 expression. A , ChIP analysis of association of Sox2 with the FGF4 or Sox2 enhancer in PARP1 +/+ and PARP1 − / − ESCs cultured under the differentiation condition. B , association of Sox2 with

    Article Snippet: We also determined the expression of FGF4 in PARP1 +/+ and PARP1 −/− cells treated with PJ34 or 3AB at different concentrations, and we found that both inhibitors caused a decrease in the mRNA level of FGF4 in differentiating PARP1 +/+ cells at a higher dosage but caused less of a decrease in PARP1 −/− cells ( C and ).

    Techniques: Expressing, Chromatin Immunoprecipitation, Cell Culture

    Association of PARP1 with Sox2 or Oct4. A , CoIP of FLAG-PARP1 and HA-Sox2 in HEK 293 cells, which were transiently cotransfected with FLAG-PARP1 and HA-Sox2 or vector. B , association of endogenous PARP1 with Sox2 or Oct4 in F9 and P19 cells. The NE of

    Journal: The Journal of Biological Chemistry

    Article Title: PARP1 Poly(ADP-ribosyl)ates Sox2 to Control Sox2 Protein Levels and FGF4 Expression during Embryonic Stem Cell Differentiation *

    doi: 10.1074/jbc.M109.033118

    Figure Lengend Snippet: Association of PARP1 with Sox2 or Oct4. A , CoIP of FLAG-PARP1 and HA-Sox2 in HEK 293 cells, which were transiently cotransfected with FLAG-PARP1 and HA-Sox2 or vector. B , association of endogenous PARP1 with Sox2 or Oct4 in F9 and P19 cells. The NE of

    Article Snippet: We also determined the expression of FGF4 in PARP1 +/+ and PARP1 −/− cells treated with PJ34 or 3AB at different concentrations, and we found that both inhibitors caused a decrease in the mRNA level of FGF4 in differentiating PARP1 +/+ cells at a higher dosage but caused less of a decrease in PARP1 −/− cells ( C and ).

    Techniques: Co-Immunoprecipitation Assay, Plasmid Preparation

    Influence of PARP-1 activity on the co-immunoprecipitation of PARP-1 by Sp1 . Nuclear proteins (300 μg) from PARP-1 +/+ cells grown either alone (control) or in the presence of hydrogen peroxide (H 2 O 2 ), PJ34 PARP-1 inhibitor, or ethidium bromide were incubated with the Sp1 Ab (sc-59) and the Sp1-protein complexes recovered by the addition of protein-A-Sepharose. The resulting immunoprecipitated proteins were then gel fractionated as in Figure 4 and Western blotted with antibodies against Sp1 or PARP-1 (C-2-10). TE: total cell extract that has not been immunoprecipitated with the Sp1 Ab. Ctl-: protein A-Sepharose added to crude nuclear proteins in the absence of Sp1 Ab and used as a negative control. IgG-Ab: normal rabbit IgG incubated with nuclear proteins prior to addition of protein A-Sepharose as a negative control.

    Journal: BMC Molecular Biology

    Article Title: Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1

    doi: 10.1186/1471-2199-8-96

    Figure Lengend Snippet: Influence of PARP-1 activity on the co-immunoprecipitation of PARP-1 by Sp1 . Nuclear proteins (300 μg) from PARP-1 +/+ cells grown either alone (control) or in the presence of hydrogen peroxide (H 2 O 2 ), PJ34 PARP-1 inhibitor, or ethidium bromide were incubated with the Sp1 Ab (sc-59) and the Sp1-protein complexes recovered by the addition of protein-A-Sepharose. The resulting immunoprecipitated proteins were then gel fractionated as in Figure 4 and Western blotted with antibodies against Sp1 or PARP-1 (C-2-10). TE: total cell extract that has not been immunoprecipitated with the Sp1 Ab. Ctl-: protein A-Sepharose added to crude nuclear proteins in the absence of Sp1 Ab and used as a negative control. IgG-Ab: normal rabbit IgG incubated with nuclear proteins prior to addition of protein A-Sepharose as a negative control.

    Article Snippet: Immunoprecipitation assay The immunoprecipitation of the Sp1 complexes was performed as follow: approximately 300 μg nuclear extracts from the PARP-1+/+ and PARP-1-/- cell lines was mixed with either the anti-Sp1 antibody (2 μg; Santa Cruz) or normal rabbit IgG (2 μg; Santa Cruz) overnight at 4°C, either alone, or in the presence of 100 μg/ml ethidium bromide [ , ], in binding buffer (10 mM Tris-HCl, 20 mM MgCl2 , 150 mM NaCl, 1 mM dithiothreitol, 10% (v/v) glycerol, 1 mM PMSF).

    Techniques: Activity Assay, Immunoprecipitation, Incubation, Western Blot, CTL Assay, Negative Control

    rPARP-1 promoter activity in PARP-1 +/+ and PARP-1 -/- cells . ( A ) The recombinant plasmids PCR3 and PCR3F2/F3/F4m were transfected into both PARP-1 +/+ and PARP-1 -/- cells grown with or without the PARP-1 inhibitor PJ34. CAT activities were measured and normalized to the amount of hGH secreted into the culture medium. Values are expressed as ((%CAT activity/100 μg proteins)/ng hGH). Asterisks (*) indicate CAT activities from cells exposed to PJ34 that are statistically different from those measured when cells are transfected with pCR3 in the absence of inhibitor whereas † corresponds to CAT activities in PARP-1 -/- cells that are statistically different from those measured in PARP-1 +/+ cells ( P

    Journal: BMC Molecular Biology

    Article Title: Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1

    doi: 10.1186/1471-2199-8-96

    Figure Lengend Snippet: rPARP-1 promoter activity in PARP-1 +/+ and PARP-1 -/- cells . ( A ) The recombinant plasmids PCR3 and PCR3F2/F3/F4m were transfected into both PARP-1 +/+ and PARP-1 -/- cells grown with or without the PARP-1 inhibitor PJ34. CAT activities were measured and normalized to the amount of hGH secreted into the culture medium. Values are expressed as ((%CAT activity/100 μg proteins)/ng hGH). Asterisks (*) indicate CAT activities from cells exposed to PJ34 that are statistically different from those measured when cells are transfected with pCR3 in the absence of inhibitor whereas † corresponds to CAT activities in PARP-1 -/- cells that are statistically different from those measured in PARP-1 +/+ cells ( P

    Article Snippet: Immunoprecipitation assay The immunoprecipitation of the Sp1 complexes was performed as follow: approximately 300 μg nuclear extracts from the PARP-1+/+ and PARP-1-/- cell lines was mixed with either the anti-Sp1 antibody (2 μg; Santa Cruz) or normal rabbit IgG (2 μg; Santa Cruz) overnight at 4°C, either alone, or in the presence of 100 μg/ml ethidium bromide [ , ], in binding buffer (10 mM Tris-HCl, 20 mM MgCl2 , 150 mM NaCl, 1 mM dithiothreitol, 10% (v/v) glycerol, 1 mM PMSF).

    Techniques: Activity Assay, Recombinant, Transfection

    In vivo influence of PARP-1 activity on the expression and DNA binding of Sp1 . ( A ) Nuclear proteins (5 μg) from PARP-1 +/+ cells grown alone (-; lane 2) or in the presence of H 2 O 2 (lane 3) or PJ34 (lane 4), added either individually or in combination (PJ34+ H 2 O 2 ; lane 5), were incubated with the Sp1 labeled probe and formation of DNA/protein complexes monitored by EMSA on a 8% native polyacrylamide gel as in Figure 2. The position of both the Sp1 and Sp3 DNA-protein complexes are shown, as well as that of the free probe (U). P: labeled probe alone (lane 1). ( B ) The extracts used in panel A were SDS-gel fractionated before being membrane-transferred and Western blotted with antibodies against Sp1 (sc-59), PARP (C-2-10) and PAR (10-H). The position of the appropriate molecular mass markers (60-, 120-, and 190 kDa) is indicated. ( C ) Nuclear proteins (5 μg) from primary cultures of HSKs grown for various periods of time (4-, 24- and 72 h) either alone (-; lanes 1, 4 and 7), or in the presence of H 2 O 2 (lanes 2, 5 and 8) or both H 2 O 2 and PJ34 (PJ34+ H 2 O 2 ; lanes 3, 6 and 9), were incubated with the Sp1 labeled probe and formation of DNA/protein complexes monitored by EMSA on a 8% native polyacrylamide gel as in panel A. ( D ) The extracts used in panel C were analyzed by Western blotting with antibodies against Sp1 (sc-59), PARP (C-2-10) and β-actin (CLT9001). Densitometric analyses of the band intensities was determined for both the Sp1 and PARP-1 proteins and normalized to that measured for β-actin. Values are shown below each corresponding track.

    Journal: BMC Molecular Biology

    Article Title: Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1

    doi: 10.1186/1471-2199-8-96

    Figure Lengend Snippet: In vivo influence of PARP-1 activity on the expression and DNA binding of Sp1 . ( A ) Nuclear proteins (5 μg) from PARP-1 +/+ cells grown alone (-; lane 2) or in the presence of H 2 O 2 (lane 3) or PJ34 (lane 4), added either individually or in combination (PJ34+ H 2 O 2 ; lane 5), were incubated with the Sp1 labeled probe and formation of DNA/protein complexes monitored by EMSA on a 8% native polyacrylamide gel as in Figure 2. The position of both the Sp1 and Sp3 DNA-protein complexes are shown, as well as that of the free probe (U). P: labeled probe alone (lane 1). ( B ) The extracts used in panel A were SDS-gel fractionated before being membrane-transferred and Western blotted with antibodies against Sp1 (sc-59), PARP (C-2-10) and PAR (10-H). The position of the appropriate molecular mass markers (60-, 120-, and 190 kDa) is indicated. ( C ) Nuclear proteins (5 μg) from primary cultures of HSKs grown for various periods of time (4-, 24- and 72 h) either alone (-; lanes 1, 4 and 7), or in the presence of H 2 O 2 (lanes 2, 5 and 8) or both H 2 O 2 and PJ34 (PJ34+ H 2 O 2 ; lanes 3, 6 and 9), were incubated with the Sp1 labeled probe and formation of DNA/protein complexes monitored by EMSA on a 8% native polyacrylamide gel as in panel A. ( D ) The extracts used in panel C were analyzed by Western blotting with antibodies against Sp1 (sc-59), PARP (C-2-10) and β-actin (CLT9001). Densitometric analyses of the band intensities was determined for both the Sp1 and PARP-1 proteins and normalized to that measured for β-actin. Values are shown below each corresponding track.

    Article Snippet: Immunoprecipitation assay The immunoprecipitation of the Sp1 complexes was performed as follow: approximately 300 μg nuclear extracts from the PARP-1+/+ and PARP-1-/- cell lines was mixed with either the anti-Sp1 antibody (2 μg; Santa Cruz) or normal rabbit IgG (2 μg; Santa Cruz) overnight at 4°C, either alone, or in the presence of 100 μg/ml ethidium bromide [ , ], in binding buffer (10 mM Tris-HCl, 20 mM MgCl2 , 150 mM NaCl, 1 mM dithiothreitol, 10% (v/v) glycerol, 1 mM PMSF).

    Techniques: In Vivo, Activity Assay, Expressing, Binding Assay, Incubation, Labeling, SDS-Gel, Western Blot

    Model of interplay between PARP-1, Sp1 and other transcription factors . ( A ) PARP-1 plays a suppressive function (indicated by 'T' bars) on the DNA binding properties of Sp1, and indirectly, on its expression as well, by the enzymatic addition of poly(ADP-ribose) units (PAR) to Sp1. PARP-1 may exert its effect by stimulating the transcriptional properties (indicated by arrows) of both AP-2 and E2F-1 by physically interacting with these transcription factors (and therefore, independently of addition of PAR), of which the latter was recognized as a component required to ensure proper transcription of the human Sp1 gene. ( B ) Once PARP-1 is stimulated by DNA damages, post-translational modification of both Sp1 and AP-2 is increased to the point that their DNA binding properties and thereby, their transcriptional capacity, is considerably decreased without significantly altering their level of expression. ( C ) However, in the absence of PARP-1, addition of PAR is abrogated and the transcriptional capacity of Sp1 becomes dramatically increased despite that its overall expression is considerably reduced primarily as a consequence of: i) a reduction in both the expression [112] and the positive transcriptional influence of E2F1 [85], a property that requires a physical interaction with PARP-1, and ii) a reduced transcriptional activity of AP-2, which also requires a physical association of this transcription factor with the middle region of PARP-1 [84]. TrC: transcriptional capacity of Sp1; Exp: level of Sp1 expression.

    Journal: BMC Molecular Biology

    Article Title: Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1

    doi: 10.1186/1471-2199-8-96

    Figure Lengend Snippet: Model of interplay between PARP-1, Sp1 and other transcription factors . ( A ) PARP-1 plays a suppressive function (indicated by 'T' bars) on the DNA binding properties of Sp1, and indirectly, on its expression as well, by the enzymatic addition of poly(ADP-ribose) units (PAR) to Sp1. PARP-1 may exert its effect by stimulating the transcriptional properties (indicated by arrows) of both AP-2 and E2F-1 by physically interacting with these transcription factors (and therefore, independently of addition of PAR), of which the latter was recognized as a component required to ensure proper transcription of the human Sp1 gene. ( B ) Once PARP-1 is stimulated by DNA damages, post-translational modification of both Sp1 and AP-2 is increased to the point that their DNA binding properties and thereby, their transcriptional capacity, is considerably decreased without significantly altering their level of expression. ( C ) However, in the absence of PARP-1, addition of PAR is abrogated and the transcriptional capacity of Sp1 becomes dramatically increased despite that its overall expression is considerably reduced primarily as a consequence of: i) a reduction in both the expression [112] and the positive transcriptional influence of E2F1 [85], a property that requires a physical interaction with PARP-1, and ii) a reduced transcriptional activity of AP-2, which also requires a physical association of this transcription factor with the middle region of PARP-1 [84]. TrC: transcriptional capacity of Sp1; Exp: level of Sp1 expression.

    Article Snippet: Immunoprecipitation assay The immunoprecipitation of the Sp1 complexes was performed as follow: approximately 300 μg nuclear extracts from the PARP-1+/+ and PARP-1-/- cell lines was mixed with either the anti-Sp1 antibody (2 μg; Santa Cruz) or normal rabbit IgG (2 μg; Santa Cruz) overnight at 4°C, either alone, or in the presence of 100 μg/ml ethidium bromide [ , ], in binding buffer (10 mM Tris-HCl, 20 mM MgCl2 , 150 mM NaCl, 1 mM dithiothreitol, 10% (v/v) glycerol, 1 mM PMSF).

    Techniques: Binding Assay, Expressing, Modification, Activity Assay

    DNA binding properties of Sp1/Sp3 and NFI in PARP-1 +/+ and PARP-1 -/- cells . ( A ) EMSA analysis of Sp1/Sp3 and NFI. Crude nuclear proteins (5 μg) from both PARP-1 +/+ and PARP-1 -/- cells were incubated with a 5' end-labeled probe bearing the high affinity binding site for either Sp1 (left) or NFI (right). Formation of DNA/protein complexes was then monitored by EMSA on an 8% (Sp1) and 10% (NFI) native polyacrylamide gel and their position revealed through autoradiography. The position of both the Sp1/Sp3 and NFI DNA-protein complexes are shown, as well as that of the free probe (U). P: labeled probe alone. ( B ) Sp1 competition experiment in EMSA. The Sp1 labeled probe used in panel A was incubated with nuclear proteins (5 μg) from both PARP-1 +/+ and PARP-1 -/- cells in the presence of either no (-) or 100- and 500-fold molar excesses of unlabeled competitor oligonucleotides (either Sp1 or NFI). Formation of DNA/protein complexes was then monitored by EMSA on an 8% native gel. ( C ) NFI competition experiment in EMSA. Same as in panel B except that the NFI double-stranded oligonucleotide was 5'-end labeled and used as probe for the assay. ( D ) Supershift experiment in EMSA. Crude nuclear proteins from both PARP-1 +/+ and PARP-1 -/- cells were incubated with the either the Sp1 (5 μg proteins were used) or NFI (10 μg proteins were used) labeled probe in the presence of either no (-), or 2 μl of a polyclonal antibody directed against Sp1 (Sp1Ab) or Sp3 (Sp3Ab) and added either individually or in combination (Sp1+Sp3Ab) (left), or with a polyclonal antibody directed against NFI (right). Formation of both the Sp1/Sp3 and NFI complexes, as well as their corresponding supershifted complexes (SSC) is indicated. P: labeled probe alone; U: unbound fraction of the labeled probe.

    Journal: BMC Molecular Biology

    Article Title: Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1

    doi: 10.1186/1471-2199-8-96

    Figure Lengend Snippet: DNA binding properties of Sp1/Sp3 and NFI in PARP-1 +/+ and PARP-1 -/- cells . ( A ) EMSA analysis of Sp1/Sp3 and NFI. Crude nuclear proteins (5 μg) from both PARP-1 +/+ and PARP-1 -/- cells were incubated with a 5' end-labeled probe bearing the high affinity binding site for either Sp1 (left) or NFI (right). Formation of DNA/protein complexes was then monitored by EMSA on an 8% (Sp1) and 10% (NFI) native polyacrylamide gel and their position revealed through autoradiography. The position of both the Sp1/Sp3 and NFI DNA-protein complexes are shown, as well as that of the free probe (U). P: labeled probe alone. ( B ) Sp1 competition experiment in EMSA. The Sp1 labeled probe used in panel A was incubated with nuclear proteins (5 μg) from both PARP-1 +/+ and PARP-1 -/- cells in the presence of either no (-) or 100- and 500-fold molar excesses of unlabeled competitor oligonucleotides (either Sp1 or NFI). Formation of DNA/protein complexes was then monitored by EMSA on an 8% native gel. ( C ) NFI competition experiment in EMSA. Same as in panel B except that the NFI double-stranded oligonucleotide was 5'-end labeled and used as probe for the assay. ( D ) Supershift experiment in EMSA. Crude nuclear proteins from both PARP-1 +/+ and PARP-1 -/- cells were incubated with the either the Sp1 (5 μg proteins were used) or NFI (10 μg proteins were used) labeled probe in the presence of either no (-), or 2 μl of a polyclonal antibody directed against Sp1 (Sp1Ab) or Sp3 (Sp3Ab) and added either individually or in combination (Sp1+Sp3Ab) (left), or with a polyclonal antibody directed against NFI (right). Formation of both the Sp1/Sp3 and NFI complexes, as well as their corresponding supershifted complexes (SSC) is indicated. P: labeled probe alone; U: unbound fraction of the labeled probe.

    Article Snippet: Immunoprecipitation assay The immunoprecipitation of the Sp1 complexes was performed as follow: approximately 300 μg nuclear extracts from the PARP-1+/+ and PARP-1-/- cell lines was mixed with either the anti-Sp1 antibody (2 μg; Santa Cruz) or normal rabbit IgG (2 μg; Santa Cruz) overnight at 4°C, either alone, or in the presence of 100 μg/ml ethidium bromide [ , ], in binding buffer (10 mM Tris-HCl, 20 mM MgCl2 , 150 mM NaCl, 1 mM dithiothreitol, 10% (v/v) glycerol, 1 mM PMSF).

    Techniques: Binding Assay, Incubation, Labeling, Autoradiography

    DNA binding and expression of the transcription factors AP-1, E2F1 and STAT-1 in PARP-1 +/+ and PARP-1 -/- cells . ( A ) EMSA analysis. Crude nuclear proteins (5 μg) from both PARP-1 +/+ and PARP-1 -/- cells were incubated with a 5' end-labeled probe bearing the high affinity binding site for AP-1, E2F1 and STAT-1. Formation of DNA/protein complexes was then monitored by EMSA on an 8% gel as detailed in Figure 2. The position of the AP-1, E2F1 and STAT-1 DNA-protein complexes is shown, as well as that of the free probe (U). P: labeled probe alone. ( B ) Coomassie blue staining of the protein samples used for conducting both the EMSA (panel A) and the Western blot experiment (panel C). One protein band present in both extract was randomly selected and its intensity determined by densitometric analysis in order to precisely calibrate the protein concentration used for the assays. ( C ) Nuclear extracts (10 μg) from both PARP-1 +/+ and PARP-1 -/- cells were examined in Western blot as in Figure 1 using antibodies directed against E2F1, STAT-1 and the AP-1 subunit c-jun. The position of the nearest molecular mass markers is indicated (60 kDa and 85 kDa).

    Journal: BMC Molecular Biology

    Article Title: Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1

    doi: 10.1186/1471-2199-8-96

    Figure Lengend Snippet: DNA binding and expression of the transcription factors AP-1, E2F1 and STAT-1 in PARP-1 +/+ and PARP-1 -/- cells . ( A ) EMSA analysis. Crude nuclear proteins (5 μg) from both PARP-1 +/+ and PARP-1 -/- cells were incubated with a 5' end-labeled probe bearing the high affinity binding site for AP-1, E2F1 and STAT-1. Formation of DNA/protein complexes was then monitored by EMSA on an 8% gel as detailed in Figure 2. The position of the AP-1, E2F1 and STAT-1 DNA-protein complexes is shown, as well as that of the free probe (U). P: labeled probe alone. ( B ) Coomassie blue staining of the protein samples used for conducting both the EMSA (panel A) and the Western blot experiment (panel C). One protein band present in both extract was randomly selected and its intensity determined by densitometric analysis in order to precisely calibrate the protein concentration used for the assays. ( C ) Nuclear extracts (10 μg) from both PARP-1 +/+ and PARP-1 -/- cells were examined in Western blot as in Figure 1 using antibodies directed against E2F1, STAT-1 and the AP-1 subunit c-jun. The position of the nearest molecular mass markers is indicated (60 kDa and 85 kDa).

    Article Snippet: Immunoprecipitation assay The immunoprecipitation of the Sp1 complexes was performed as follow: approximately 300 μg nuclear extracts from the PARP-1+/+ and PARP-1-/- cell lines was mixed with either the anti-Sp1 antibody (2 μg; Santa Cruz) or normal rabbit IgG (2 μg; Santa Cruz) overnight at 4°C, either alone, or in the presence of 100 μg/ml ethidium bromide [ , ], in binding buffer (10 mM Tris-HCl, 20 mM MgCl2 , 150 mM NaCl, 1 mM dithiothreitol, 10% (v/v) glycerol, 1 mM PMSF).

    Techniques: Binding Assay, Expressing, Incubation, Labeling, Staining, Western Blot, Protein Concentration

    Co-immunoprecipitation of Sp1 and PARP-1 in protein extracts from PARP-1 +/+ and PARP-1 -/- cells . ( A ) Immunoprecipitation of the Sp1-protein complexes in PARP-1 +/+ and PARP-1 -/- nuclear extracts. Crude nuclear proteins (300 μg) from both PARP-1 +/+ and PARP-1 -/- cells were incubated with the Sp1 Ab (sc-59) and the Sp1-protein complexes recovered by the addition of protein-A-Sepharose. The resulting immunoprecipitated proteins were then SDS-gel fractionated before being membrane-transferred and Western blotted with antibodies against Sp1, PARP-1 (C-2-10) and PAR (LP-9610). Ctl-: protein A-Sepharose added to crude nuclear proteins in the absence of Sp1 Ab and used as a negative control. IgG-Ab: normal rabbit IgG incubated with nuclear proteins prior to addition of protein A-Sepharose as a negative control. ( B ) Immunoprecipitation of the PARP-1-protein complexes in PARP-1 +/+ and PARP-1 -/- nuclear extracts. Same as in panel A except that the immunoprecipitation was conducted using the PARP-1 F-123 Ab. The blotted, PARP-1-immunoprecipitated proteins were then analyzed with the PARP-1 (422), Sp1 (sc-59), Sp3 (sc-644), and PAR (LP-9610) antibodies. Negative controls (Ctl- and IgG-Ab) are as in panel A. TE: total cell extract that has not been immunoprecipitated with the PARP-1 Ab.

    Journal: BMC Molecular Biology

    Article Title: Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1

    doi: 10.1186/1471-2199-8-96

    Figure Lengend Snippet: Co-immunoprecipitation of Sp1 and PARP-1 in protein extracts from PARP-1 +/+ and PARP-1 -/- cells . ( A ) Immunoprecipitation of the Sp1-protein complexes in PARP-1 +/+ and PARP-1 -/- nuclear extracts. Crude nuclear proteins (300 μg) from both PARP-1 +/+ and PARP-1 -/- cells were incubated with the Sp1 Ab (sc-59) and the Sp1-protein complexes recovered by the addition of protein-A-Sepharose. The resulting immunoprecipitated proteins were then SDS-gel fractionated before being membrane-transferred and Western blotted with antibodies against Sp1, PARP-1 (C-2-10) and PAR (LP-9610). Ctl-: protein A-Sepharose added to crude nuclear proteins in the absence of Sp1 Ab and used as a negative control. IgG-Ab: normal rabbit IgG incubated with nuclear proteins prior to addition of protein A-Sepharose as a negative control. ( B ) Immunoprecipitation of the PARP-1-protein complexes in PARP-1 +/+ and PARP-1 -/- nuclear extracts. Same as in panel A except that the immunoprecipitation was conducted using the PARP-1 F-123 Ab. The blotted, PARP-1-immunoprecipitated proteins were then analyzed with the PARP-1 (422), Sp1 (sc-59), Sp3 (sc-644), and PAR (LP-9610) antibodies. Negative controls (Ctl- and IgG-Ab) are as in panel A. TE: total cell extract that has not been immunoprecipitated with the PARP-1 Ab.

    Article Snippet: Immunoprecipitation assay The immunoprecipitation of the Sp1 complexes was performed as follow: approximately 300 μg nuclear extracts from the PARP-1+/+ and PARP-1-/- cell lines was mixed with either the anti-Sp1 antibody (2 μg; Santa Cruz) or normal rabbit IgG (2 μg; Santa Cruz) overnight at 4°C, either alone, or in the presence of 100 μg/ml ethidium bromide [ , ], in binding buffer (10 mM Tris-HCl, 20 mM MgCl2 , 150 mM NaCl, 1 mM dithiothreitol, 10% (v/v) glycerol, 1 mM PMSF).

    Techniques: Immunoprecipitation, Incubation, SDS-Gel, Western Blot, CTL Assay, Negative Control

    PARP-1-dependent poly(ADP-ribosyl)ation of Sp1 in vitro . ( A ) Recombinant Sp1 protein was incubated in reaction buffer either alone (lane 4) or with purified bovine PARP-1 (1 unit) in the presence of 200 μM NAD+ (lane 5). The reaction mixture was subjected to Western blot analysis with the PARP-1 (C-2-10), Sp1 (sc-59) and PAR (LP-9610) antibodies. When indicated, the PARP inhibitor PJ34 was added to the reaction mixture with purified PARP-1 alone (lane 3) or in the presence of recombinant Sp1 (lane 6). When indicated, samples from the in vitro PARP assay were electrophoresed and electrotransfered onto nitrocellulose membranes. The PAR covalently linked onto the automodified PARP-1 and Sp1 proteins was then erased by incubation with PARG and the proteins analyzed by Western blotting with the same antibodies as detailed above (lane 8). Lane 1: PARP-1 alone; lane 2: PARP-1 incubated with NAD+; lane 3: same as in lane 2 plus PJ34; lane 7: same as in lane 5 but incubated in PARG buffer without addition of PARG-1. The position of modified PARP-1 (PARP-1Mod) and Sp1 (Sp1Mod) is indicated (left) along with the appropriate molecular mass marker (right). ( B ) Recombinant Sp1 was incubated in reaction buffer containing 200 μM NAD+ and nicked DNA either alone (+SP1; lane 3) or with purified bovine PARP-1 (1 unit) (+Sp1/PARP-1; lane 4). A sample (16 μl) from the reaction mixture was then incubated with the 5'-end labeled Sp1 oligonucleotide and formation of DNA-protein complexes monitored by EMSA as in Figure 2. As a control, the PARP-1 inhibitor PJ34 was added to the reaction mixture containing PARP-1/NAD + /Sp1 (+Sp1/PARP-1/PJ34; lane 5). Lane 1: labeled probe alone in reaction mix (P); Lane 2: labeled probe incubated in buffer D with PARP-1 but in the absence of NAD and Sp1 (+PARP-1). The position of both the Sp1 complex (Sp1) and the free probe (U) is indicated.

    Journal: BMC Molecular Biology

    Article Title: Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1

    doi: 10.1186/1471-2199-8-96

    Figure Lengend Snippet: PARP-1-dependent poly(ADP-ribosyl)ation of Sp1 in vitro . ( A ) Recombinant Sp1 protein was incubated in reaction buffer either alone (lane 4) or with purified bovine PARP-1 (1 unit) in the presence of 200 μM NAD+ (lane 5). The reaction mixture was subjected to Western blot analysis with the PARP-1 (C-2-10), Sp1 (sc-59) and PAR (LP-9610) antibodies. When indicated, the PARP inhibitor PJ34 was added to the reaction mixture with purified PARP-1 alone (lane 3) or in the presence of recombinant Sp1 (lane 6). When indicated, samples from the in vitro PARP assay were electrophoresed and electrotransfered onto nitrocellulose membranes. The PAR covalently linked onto the automodified PARP-1 and Sp1 proteins was then erased by incubation with PARG and the proteins analyzed by Western blotting with the same antibodies as detailed above (lane 8). Lane 1: PARP-1 alone; lane 2: PARP-1 incubated with NAD+; lane 3: same as in lane 2 plus PJ34; lane 7: same as in lane 5 but incubated in PARG buffer without addition of PARG-1. The position of modified PARP-1 (PARP-1Mod) and Sp1 (Sp1Mod) is indicated (left) along with the appropriate molecular mass marker (right). ( B ) Recombinant Sp1 was incubated in reaction buffer containing 200 μM NAD+ and nicked DNA either alone (+SP1; lane 3) or with purified bovine PARP-1 (1 unit) (+Sp1/PARP-1; lane 4). A sample (16 μl) from the reaction mixture was then incubated with the 5'-end labeled Sp1 oligonucleotide and formation of DNA-protein complexes monitored by EMSA as in Figure 2. As a control, the PARP-1 inhibitor PJ34 was added to the reaction mixture containing PARP-1/NAD + /Sp1 (+Sp1/PARP-1/PJ34; lane 5). Lane 1: labeled probe alone in reaction mix (P); Lane 2: labeled probe incubated in buffer D with PARP-1 but in the absence of NAD and Sp1 (+PARP-1). The position of both the Sp1 complex (Sp1) and the free probe (U) is indicated.

    Article Snippet: Immunoprecipitation assay The immunoprecipitation of the Sp1 complexes was performed as follow: approximately 300 μg nuclear extracts from the PARP-1+/+ and PARP-1-/- cell lines was mixed with either the anti-Sp1 antibody (2 μg; Santa Cruz) or normal rabbit IgG (2 μg; Santa Cruz) overnight at 4°C, either alone, or in the presence of 100 μg/ml ethidium bromide [ , ], in binding buffer (10 mM Tris-HCl, 20 mM MgCl2 , 150 mM NaCl, 1 mM dithiothreitol, 10% (v/v) glycerol, 1 mM PMSF).

    Techniques: In Vitro, Recombinant, Incubation, Purification, Western Blot, Modification, Marker, Labeling

    Expression of PARP-1, Sp1, Sp3 and NFI in PARP-1 +/+ and PARP-1 -/- cells . Crude nuclear extracts (10 μg) from both PARP-1 +/+ and PARP-1 -/- cells were examined in Western blot using antibodies directed against PARP-1, Sp1, Sp3 and NFI. The position of the 120 kDa and 60 kDa proteins used as molecular mass markers is indicated. The asterisk indicates the position of the typical NFI complex whereas the arrowhead designates NFI complexes with a reduced electrophoretic mobility that predominated in the extract from PARP-1 -/- cells. Data of one from three similar experiments are presented.

    Journal: BMC Molecular Biology

    Article Title: Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1

    doi: 10.1186/1471-2199-8-96

    Figure Lengend Snippet: Expression of PARP-1, Sp1, Sp3 and NFI in PARP-1 +/+ and PARP-1 -/- cells . Crude nuclear extracts (10 μg) from both PARP-1 +/+ and PARP-1 -/- cells were examined in Western blot using antibodies directed against PARP-1, Sp1, Sp3 and NFI. The position of the 120 kDa and 60 kDa proteins used as molecular mass markers is indicated. The asterisk indicates the position of the typical NFI complex whereas the arrowhead designates NFI complexes with a reduced electrophoretic mobility that predominated in the extract from PARP-1 -/- cells. Data of one from three similar experiments are presented.

    Article Snippet: Immunoprecipitation assay The immunoprecipitation of the Sp1 complexes was performed as follow: approximately 300 μg nuclear extracts from the PARP-1+/+ and PARP-1-/- cell lines was mixed with either the anti-Sp1 antibody (2 μg; Santa Cruz) or normal rabbit IgG (2 μg; Santa Cruz) overnight at 4°C, either alone, or in the presence of 100 μg/ml ethidium bromide [ , ], in binding buffer (10 mM Tris-HCl, 20 mM MgCl2 , 150 mM NaCl, 1 mM dithiothreitol, 10% (v/v) glycerol, 1 mM PMSF).

    Techniques: Expressing, Western Blot

    RNF146 overexpression is cytoprotective in transiently transfected cells. (A, B) 293T cells were transiently transfected with CMV-RNF146 vector (CMV-RNF) or empty pcDNA3.1 + vector (CTL) and exposed to H 2 O 2 to induce PARP-1 activation and cell death. (A) Cell viability was measured after 3 h by the MTT assay. (B) PARP-1 activation is measured at its the peak (5 min in 293T cells) by immunoblotting for poly(ADP-ribose) (PAR) polymer at 116 kDa. The PARP inhibitor PJ34 (3 μmol/L) was used in pretreatment (30 min). Representative blot image and densitometric analysis results are shown. (C) H9c2 cells were transiently transfected with pcDNA-RNF146 and exposed to H 2 O 2 (600 μmol/L, 30 min, peak of PARP activation in H9c2 cells). Cells were fixed and immunostained for RNF146 (left panel) and PAR polymer (right panel). Decreased PAR signal was detectable (right panel, arrow) in cells showing RNF146 overexpression (left panel, arrow).

    Journal: Molecular Medicine

    Article Title: Modulation of Poly(ADP-Ribose) Polymerase-1 (PARP-1)-Mediated Oxidative Cell Injury by Ring Finger Protein 146 (RNF146) in Cardiac Myocytes

    doi: 10.2119/molmed.2014.00102

    Figure Lengend Snippet: RNF146 overexpression is cytoprotective in transiently transfected cells. (A, B) 293T cells were transiently transfected with CMV-RNF146 vector (CMV-RNF) or empty pcDNA3.1 + vector (CTL) and exposed to H 2 O 2 to induce PARP-1 activation and cell death. (A) Cell viability was measured after 3 h by the MTT assay. (B) PARP-1 activation is measured at its the peak (5 min in 293T cells) by immunoblotting for poly(ADP-ribose) (PAR) polymer at 116 kDa. The PARP inhibitor PJ34 (3 μmol/L) was used in pretreatment (30 min). Representative blot image and densitometric analysis results are shown. (C) H9c2 cells were transiently transfected with pcDNA-RNF146 and exposed to H 2 O 2 (600 μmol/L, 30 min, peak of PARP activation in H9c2 cells). Cells were fixed and immunostained for RNF146 (left panel) and PAR polymer (right panel). Decreased PAR signal was detectable (right panel, arrow) in cells showing RNF146 overexpression (left panel, arrow).

    Article Snippet: Cells were permeabilized with 0.2% Triton X-100, blocked with 2.5% horse serum (Vector Laboratories, Burlingame, CA, USA) and probed with PARP-1 (1:100; Cell Signaling Technology) or RNF-146 antibody (1:100, Abnova, Walnut, CA) followed by incubation with Alexa Fluor 546–labeled secondary antibody (Invitrogen/Life Technologies).

    Techniques: Over Expression, Transfection, Plasmid Preparation, CTL Assay, Activation Assay, MTT Assay

    Ischemia-reperfusion injury induces the cytoplasmic release of PARP-1 and the nuclear translocation of RNF146 in vivo . (A, B) Regional ischemia-reperfusion injury was induced in mouse hearts by occlusion of the LAD coronary artery for 30 min; then the ligature was removed and the hearts were reperfused for 2 h. The ischemic and nonischemic areas of hearts subjected to ischemia-reperfusion (I/R) injury and sham-operated (CTL) hearts were fixed in formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin (HE), Masson trichrome stain and immunostained with PARP-1, PAR or RNF146 antibodies. (A) Mild structural changes are detectable on HE- and trichrome-stained sections. PARP-1 immunostaining shows decreased nuclear staining with areas of extranuclear positivity (arrows). PARP-1 activation was detectable as nuclear positivity with PAR antibody (arrows). Nuclear translocation of RNF146 (marked with arrows) and diminished cytoplasmic staining was detectable. (B) Nuclear positivity was evaluated on five areas of each immunostained section and expressed as percentage values of PARP-1, PAR and RNF146 positivity (n = 5/group, * p

    Journal: Molecular Medicine

    Article Title: Modulation of Poly(ADP-Ribose) Polymerase-1 (PARP-1)-Mediated Oxidative Cell Injury by Ring Finger Protein 146 (RNF146) in Cardiac Myocytes

    doi: 10.2119/molmed.2014.00102

    Figure Lengend Snippet: Ischemia-reperfusion injury induces the cytoplasmic release of PARP-1 and the nuclear translocation of RNF146 in vivo . (A, B) Regional ischemia-reperfusion injury was induced in mouse hearts by occlusion of the LAD coronary artery for 30 min; then the ligature was removed and the hearts were reperfused for 2 h. The ischemic and nonischemic areas of hearts subjected to ischemia-reperfusion (I/R) injury and sham-operated (CTL) hearts were fixed in formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin (HE), Masson trichrome stain and immunostained with PARP-1, PAR or RNF146 antibodies. (A) Mild structural changes are detectable on HE- and trichrome-stained sections. PARP-1 immunostaining shows decreased nuclear staining with areas of extranuclear positivity (arrows). PARP-1 activation was detectable as nuclear positivity with PAR antibody (arrows). Nuclear translocation of RNF146 (marked with arrows) and diminished cytoplasmic staining was detectable. (B) Nuclear positivity was evaluated on five areas of each immunostained section and expressed as percentage values of PARP-1, PAR and RNF146 positivity (n = 5/group, * p

    Article Snippet: Cells were permeabilized with 0.2% Triton X-100, blocked with 2.5% horse serum (Vector Laboratories, Burlingame, CA, USA) and probed with PARP-1 (1:100; Cell Signaling Technology) or RNF-146 antibody (1:100, Abnova, Walnut, CA) followed by incubation with Alexa Fluor 546–labeled secondary antibody (Invitrogen/Life Technologies).

    Techniques: Translocation Assay, In Vivo, CTL Assay, Staining, Immunostaining, Activation Assay

    Ischemia-reperfusion injury results in an overall decrease in RNF146 and PARP-1 content in the myocardial tissue in vivo . (A–C) Ischemia-reperfusion (I/R) injury was induced in mouse hearts by LAD coronary artery occlusion for 30 min, followed by a 3-h reperfusion. Levels of PARP-1, RNF146, free ubiquitin and HSP70 were measured by Western blotting in the ischemic and nonischemic areas of I/R hearts and sham-operated (CTL) hearts. (A) Representative blot images and respective actin normalization signals are shown. (B) Densitometric analysis results of normalized values of PARP-1, RNF-146, free ubiquitin and HSP-70 are shown. n = 4/group. * p

    Journal: Molecular Medicine

    Article Title: Modulation of Poly(ADP-Ribose) Polymerase-1 (PARP-1)-Mediated Oxidative Cell Injury by Ring Finger Protein 146 (RNF146) in Cardiac Myocytes

    doi: 10.2119/molmed.2014.00102

    Figure Lengend Snippet: Ischemia-reperfusion injury results in an overall decrease in RNF146 and PARP-1 content in the myocardial tissue in vivo . (A–C) Ischemia-reperfusion (I/R) injury was induced in mouse hearts by LAD coronary artery occlusion for 30 min, followed by a 3-h reperfusion. Levels of PARP-1, RNF146, free ubiquitin and HSP70 were measured by Western blotting in the ischemic and nonischemic areas of I/R hearts and sham-operated (CTL) hearts. (A) Representative blot images and respective actin normalization signals are shown. (B) Densitometric analysis results of normalized values of PARP-1, RNF-146, free ubiquitin and HSP-70 are shown. n = 4/group. * p

    Article Snippet: Cells were permeabilized with 0.2% Triton X-100, blocked with 2.5% horse serum (Vector Laboratories, Burlingame, CA, USA) and probed with PARP-1 (1:100; Cell Signaling Technology) or RNF-146 antibody (1:100, Abnova, Walnut, CA) followed by incubation with Alexa Fluor 546–labeled secondary antibody (Invitrogen/Life Technologies).

    Techniques: In Vivo, Western Blot, CTL Assay

    PARP-1 release from the nucleus coincides with increased RNF146 expression during mitosis. (A) H9c2 cells were immunostained with PARP-1 and Alexa Fluor 546 secondary antibodies (red); nuclei were labeled with Hoechst 33342 (blue) and actin with Alexa Fluor 488 phalloidin (green). Mitotic phases are shown with merged Hoechst and PARP-1 channels; dividing cells are labeled with arrows. Intense cytoplasmic PARP-1 staining is observed from prometaphase (when the nuclear membrane disintegrates) to anaphase. PARP-1 staining intensity decreases by the telophase (when new nuclear membrane is formed) in the two daughter cells (connected arrows). (B) H9c2 cells were immunostained with RNF146 and Alexa Fluor 546 secondary antibodies (red); nuclei were labeled with Hoechst 33342 (blue) and actin with Alexa Fluor 488 phalloidin (green). Mitotic phases are shown with merged Hoechst and RNF146 channels; dividing cells are labeled with arrows. RNF146 staining intensity is increased in dividing cells from prometaphase to anaphase. RNF146 staining intensity decreases in the two daughter cells (connected arrows) in the telophase.

    Journal: Molecular Medicine

    Article Title: Modulation of Poly(ADP-Ribose) Polymerase-1 (PARP-1)-Mediated Oxidative Cell Injury by Ring Finger Protein 146 (RNF146) in Cardiac Myocytes

    doi: 10.2119/molmed.2014.00102

    Figure Lengend Snippet: PARP-1 release from the nucleus coincides with increased RNF146 expression during mitosis. (A) H9c2 cells were immunostained with PARP-1 and Alexa Fluor 546 secondary antibodies (red); nuclei were labeled with Hoechst 33342 (blue) and actin with Alexa Fluor 488 phalloidin (green). Mitotic phases are shown with merged Hoechst and PARP-1 channels; dividing cells are labeled with arrows. Intense cytoplasmic PARP-1 staining is observed from prometaphase (when the nuclear membrane disintegrates) to anaphase. PARP-1 staining intensity decreases by the telophase (when new nuclear membrane is formed) in the two daughter cells (connected arrows). (B) H9c2 cells were immunostained with RNF146 and Alexa Fluor 546 secondary antibodies (red); nuclei were labeled with Hoechst 33342 (blue) and actin with Alexa Fluor 488 phalloidin (green). Mitotic phases are shown with merged Hoechst and RNF146 channels; dividing cells are labeled with arrows. RNF146 staining intensity is increased in dividing cells from prometaphase to anaphase. RNF146 staining intensity decreases in the two daughter cells (connected arrows) in the telophase.

    Article Snippet: Cells were permeabilized with 0.2% Triton X-100, blocked with 2.5% horse serum (Vector Laboratories, Burlingame, CA, USA) and probed with PARP-1 (1:100; Cell Signaling Technology) or RNF-146 antibody (1:100, Abnova, Walnut, CA) followed by incubation with Alexa Fluor 546–labeled secondary antibody (Invitrogen/Life Technologies).

    Techniques: Expressing, Labeling, Staining

    RNF146 directly interacts with PARP-1. (A) Nuclear translocation of RNF146 is detected in H9c2 cardiomyoblasts exposed to H 2 O 2 (600 μmol/L, 30 min). The cells were fixed in formalin, stained with RNF146 antibody and visualized with Alexa Fluor 488–labeled secondary antibody. Resting cells show cytoplasmic staining, whereas intense nuclear staining is detectable in cells exposed to H 2 O 2 . (B, C) H9c2 cells stably transfected with RNF146 (CMV-RNF146) or with control vector (CTL) were exposed to H 2 O 2 (1 mmol/L, 30 min). (B) Immunoprecipitation (IP) was performed with PARP-1 or RNF146 antibodies, and the presence of PARP-1 and RNF146 proteins and activation of PARP-1 (PARylation at 116 kDa, PAR) were tested by Western blotting. Interaction of RNF146 and PARP-1 (both PARylated and non-PARylated forms) was detected. (C) Immunoprecipitation by PARP-1 and ubiquitin antibodies was followed by Western blotting for PARP-1 and ubiquitin and revealed PARP-1 ubiquitination and the consumption of free ubiquitin.

    Journal: Molecular Medicine

    Article Title: Modulation of Poly(ADP-Ribose) Polymerase-1 (PARP-1)-Mediated Oxidative Cell Injury by Ring Finger Protein 146 (RNF146) in Cardiac Myocytes

    doi: 10.2119/molmed.2014.00102

    Figure Lengend Snippet: RNF146 directly interacts with PARP-1. (A) Nuclear translocation of RNF146 is detected in H9c2 cardiomyoblasts exposed to H 2 O 2 (600 μmol/L, 30 min). The cells were fixed in formalin, stained with RNF146 antibody and visualized with Alexa Fluor 488–labeled secondary antibody. Resting cells show cytoplasmic staining, whereas intense nuclear staining is detectable in cells exposed to H 2 O 2 . (B, C) H9c2 cells stably transfected with RNF146 (CMV-RNF146) or with control vector (CTL) were exposed to H 2 O 2 (1 mmol/L, 30 min). (B) Immunoprecipitation (IP) was performed with PARP-1 or RNF146 antibodies, and the presence of PARP-1 and RNF146 proteins and activation of PARP-1 (PARylation at 116 kDa, PAR) were tested by Western blotting. Interaction of RNF146 and PARP-1 (both PARylated and non-PARylated forms) was detected. (C) Immunoprecipitation by PARP-1 and ubiquitin antibodies was followed by Western blotting for PARP-1 and ubiquitin and revealed PARP-1 ubiquitination and the consumption of free ubiquitin.

    Article Snippet: Cells were permeabilized with 0.2% Triton X-100, blocked with 2.5% horse serum (Vector Laboratories, Burlingame, CA, USA) and probed with PARP-1 (1:100; Cell Signaling Technology) or RNF-146 antibody (1:100, Abnova, Walnut, CA) followed by incubation with Alexa Fluor 546–labeled secondary antibody (Invitrogen/Life Technologies).

    Techniques: Translocation Assay, Staining, Labeling, Stable Transfection, Transfection, Plasmid Preparation, CTL Assay, Immunoprecipitation, Activation Assay, Western Blot

    Oxidative stress decreases the levels of RNF146 and PARP-1 proteins and induces nuclear-to-cytoplasmatic PARP-1 release. (A, B) H9c2 cardiomyocytes stably transfected with CMV-RNF146 (CMV-RNF) or transfected with RNF146 siRNA (siRNF) and exposed to H 2 O 2 (1 mmol/L, 30 min). PARP-1 activation (PARylation of PARP-1 at 116kDa, PAR) and the expression of RNF146 were measured by Western blotting. Representative blots (A) and results of densitometric analysis (B) are shown. (C) PARP-1 expression was measured in H9c2 cells after H 2 O 2 injury (300 or 600 μmol/L) at 4, 8, 16 or 24 h. Immunoblots and densitometric analysis results are shown. * p

    Journal: Molecular Medicine

    Article Title: Modulation of Poly(ADP-Ribose) Polymerase-1 (PARP-1)-Mediated Oxidative Cell Injury by Ring Finger Protein 146 (RNF146) in Cardiac Myocytes

    doi: 10.2119/molmed.2014.00102

    Figure Lengend Snippet: Oxidative stress decreases the levels of RNF146 and PARP-1 proteins and induces nuclear-to-cytoplasmatic PARP-1 release. (A, B) H9c2 cardiomyocytes stably transfected with CMV-RNF146 (CMV-RNF) or transfected with RNF146 siRNA (siRNF) and exposed to H 2 O 2 (1 mmol/L, 30 min). PARP-1 activation (PARylation of PARP-1 at 116kDa, PAR) and the expression of RNF146 were measured by Western blotting. Representative blots (A) and results of densitometric analysis (B) are shown. (C) PARP-1 expression was measured in H9c2 cells after H 2 O 2 injury (300 or 600 μmol/L) at 4, 8, 16 or 24 h. Immunoblots and densitometric analysis results are shown. * p

    Article Snippet: Cells were permeabilized with 0.2% Triton X-100, blocked with 2.5% horse serum (Vector Laboratories, Burlingame, CA, USA) and probed with PARP-1 (1:100; Cell Signaling Technology) or RNF-146 antibody (1:100, Abnova, Walnut, CA) followed by incubation with Alexa Fluor 546–labeled secondary antibody (Invitrogen/Life Technologies).

    Techniques: Stable Transfection, Transfection, Activation Assay, Expressing, Western Blot

    RNF146 controls PARP-1 degradation in oxidative stress. PARP-1 activation is triggered by DNA strand breaks in oxidative stress. The enzyme possesses high catalytic activity, builds large poly(ADP-ribose) (PAR) polymers on target proteins and can deplete the cellular NAD + ). The major enzyme responsible for PAR catabolism is the cytoplasmic poly(ADP-ribose) glycohydrolase (PARG), which needs to translocate to the nucleus to access the PAR polymers. RNF146 is a protein that is normally cytoplasmatic (where it can capture PARylated proteins; based on the current results, during ischemia, PARP-1 can be translocated into the cytoplasm, and it can act as one of its interacting proteins). Moreover, the current results show that it can also rapidly translocate into the nucleus to directly interact with PARP-1. As an E3-ubiquitin ligase, RNF146 promotes the rapid proteasomal degradation of PARP-1. Thus, it irreversibly inactivates PARP-1. We hypothesize that this process may serve as a protective mechanism to limit the cellular/mitochondrial dysfunction induced by PARP-1 overactivation.

    Journal: Molecular Medicine

    Article Title: Modulation of Poly(ADP-Ribose) Polymerase-1 (PARP-1)-Mediated Oxidative Cell Injury by Ring Finger Protein 146 (RNF146) in Cardiac Myocytes

    doi: 10.2119/molmed.2014.00102

    Figure Lengend Snippet: RNF146 controls PARP-1 degradation in oxidative stress. PARP-1 activation is triggered by DNA strand breaks in oxidative stress. The enzyme possesses high catalytic activity, builds large poly(ADP-ribose) (PAR) polymers on target proteins and can deplete the cellular NAD + ). The major enzyme responsible for PAR catabolism is the cytoplasmic poly(ADP-ribose) glycohydrolase (PARG), which needs to translocate to the nucleus to access the PAR polymers. RNF146 is a protein that is normally cytoplasmatic (where it can capture PARylated proteins; based on the current results, during ischemia, PARP-1 can be translocated into the cytoplasm, and it can act as one of its interacting proteins). Moreover, the current results show that it can also rapidly translocate into the nucleus to directly interact with PARP-1. As an E3-ubiquitin ligase, RNF146 promotes the rapid proteasomal degradation of PARP-1. Thus, it irreversibly inactivates PARP-1. We hypothesize that this process may serve as a protective mechanism to limit the cellular/mitochondrial dysfunction induced by PARP-1 overactivation.

    Article Snippet: Cells were permeabilized with 0.2% Triton X-100, blocked with 2.5% horse serum (Vector Laboratories, Burlingame, CA, USA) and probed with PARP-1 (1:100; Cell Signaling Technology) or RNF-146 antibody (1:100, Abnova, Walnut, CA) followed by incubation with Alexa Fluor 546–labeled secondary antibody (Invitrogen/Life Technologies).

    Techniques: Activation Assay, Activity Assay, Activated Clotting Time Assay

    PARP-1 is released from the nucleus during oxidative stress: enhancement by RNF146 overexpression. (A, B) RNF146 was overexpressed in the cells by stable transfection with pcDNA-RNF146 (CMV-RNF146). Control cells (CTL) were simultaneously transfected with β-galactosidase expression vector and selected with G418. (A) H9c2 cells were exposed to H 2 O 2 (1 mmol/L, 30 min) and fixed by neutral buffered formalin. PARP-1 immunostaining was visualized using Alexa Fluor 546 (red). Actin filaments were stained with Alexa Fluor 488 phalloidin (green) and the nucleus with Hoechst 33342 (blue). Left panel shows control cells and right panel shows cells exposed to H 2 O 2 . All three channels are merged on the top panels; the PARP-1 and phalloidin channels are merged in the middle ones and PARP-1 and Hoechst are merged on the bottom panels. (B) RNF146 and PARP-1 expression was measured by Western blotting in stably transfected cells and the signal was normalized to actin signal. Representative blot image and densitometric analysis results are shown; * p

    Journal: Molecular Medicine

    Article Title: Modulation of Poly(ADP-Ribose) Polymerase-1 (PARP-1)-Mediated Oxidative Cell Injury by Ring Finger Protein 146 (RNF146) in Cardiac Myocytes

    doi: 10.2119/molmed.2014.00102

    Figure Lengend Snippet: PARP-1 is released from the nucleus during oxidative stress: enhancement by RNF146 overexpression. (A, B) RNF146 was overexpressed in the cells by stable transfection with pcDNA-RNF146 (CMV-RNF146). Control cells (CTL) were simultaneously transfected with β-galactosidase expression vector and selected with G418. (A) H9c2 cells were exposed to H 2 O 2 (1 mmol/L, 30 min) and fixed by neutral buffered formalin. PARP-1 immunostaining was visualized using Alexa Fluor 546 (red). Actin filaments were stained with Alexa Fluor 488 phalloidin (green) and the nucleus with Hoechst 33342 (blue). Left panel shows control cells and right panel shows cells exposed to H 2 O 2 . All three channels are merged on the top panels; the PARP-1 and phalloidin channels are merged in the middle ones and PARP-1 and Hoechst are merged on the bottom panels. (B) RNF146 and PARP-1 expression was measured by Western blotting in stably transfected cells and the signal was normalized to actin signal. Representative blot image and densitometric analysis results are shown; * p

    Article Snippet: Cells were permeabilized with 0.2% Triton X-100, blocked with 2.5% horse serum (Vector Laboratories, Burlingame, CA, USA) and probed with PARP-1 (1:100; Cell Signaling Technology) or RNF-146 antibody (1:100, Abnova, Walnut, CA) followed by incubation with Alexa Fluor 546–labeled secondary antibody (Invitrogen/Life Technologies).

    Techniques: Over Expression, Stable Transfection, CTL Assay, Transfection, Expressing, Plasmid Preparation, Immunostaining, Staining, Western Blot

    Biochemical and cellular characteristics of natural PARP1 variants. ( A ) An inherited PARP1 mutation identified by exome sequencing in a patient with pediatric colorectal cancer. The panel on the left shows a subset of the sequencing reads spanning the individual mutations (data based on hg19); the panel on the right shows the validation by Sanger sequencing in the child and the maternal samples to indicate the mode of inheritance. Position of the mutation is indicated by red arrows. ( B ) Biochemical characterization of natural PARP1 variants as used in this study. Rec. enzymes were expressed in the Sf 9/baculovirus system and purified via size exclusion and affinity chromatography. PARP1 activity was examined by incubating 5 nM PARP1 with increasing concentrations of NAD + as indicated in a reaction mixture as described in material and methods section. Afterward, 15% of reaction mixtures were slot-blotted on a nylon membrane (see Supplementary Figure S7) and PAR content was analyzed by immunochemical staining using the 10H antibody. Means of n = 3 independent experiments. A non-linear Michaelis-Menten model was used for curve fit. Statistical analysis using 2-way ANOVA testing. ( C ) Analysis of intracellular PARP1 activity in PARP1 KO cells reconstituted with PARP1 variants as indicated 2 days after transfection by immuno-epifluorescence microscopy as shown in Figure 4 (for representative raw data refer to Supplementary Figure S7). Cells were treated with H 2 O 2 for 5 min in concentrations as indicated, and PAR levels of eGFP-positive cells were examined using the anti-PAR-specific mAB 10H. Means ± SEM of n = 5 independent experiments. Statistical analysis was performed using matched two-way ANOVA testing and Sidak's post-test. ( D ) Time-course analysis of PAR formation in PARP1-reconstituted cells after treatment of cells with 250 μM H 2 O 2 . Means ± SEM of n = 4 independent experiments ( > 100 cells per experiment). Statistical analysis using matched two-way ANOVA testing and Sidak's post-test. ( E ) NAD + levels in WT, PARP1 KO and PARP1-reconstituted cells ± H 2 O 2 treatment for 7 min as evaluated by an enzymatic NAD + cycling assay. Means ± SEM of n = 3 independent experiments. Statistical analysis was performed via 2-way ANOVA testing and Sidak's post-test. ( F ) Recruitment and dissociation kinetics of natural PARP1 variants at sites of laser-induced DNA damage. For representative raw data refer to Supplementary Figure S7. Means ± SEM. Evaluation from ≥35 cells from three independent experiments. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test.

    Journal: Nucleic Acids Research

    Article Title: Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells

    doi: 10.1093/nar/gkw859

    Figure Lengend Snippet: Biochemical and cellular characteristics of natural PARP1 variants. ( A ) An inherited PARP1 mutation identified by exome sequencing in a patient with pediatric colorectal cancer. The panel on the left shows a subset of the sequencing reads spanning the individual mutations (data based on hg19); the panel on the right shows the validation by Sanger sequencing in the child and the maternal samples to indicate the mode of inheritance. Position of the mutation is indicated by red arrows. ( B ) Biochemical characterization of natural PARP1 variants as used in this study. Rec. enzymes were expressed in the Sf 9/baculovirus system and purified via size exclusion and affinity chromatography. PARP1 activity was examined by incubating 5 nM PARP1 with increasing concentrations of NAD + as indicated in a reaction mixture as described in material and methods section. Afterward, 15% of reaction mixtures were slot-blotted on a nylon membrane (see Supplementary Figure S7) and PAR content was analyzed by immunochemical staining using the 10H antibody. Means of n = 3 independent experiments. A non-linear Michaelis-Menten model was used for curve fit. Statistical analysis using 2-way ANOVA testing. ( C ) Analysis of intracellular PARP1 activity in PARP1 KO cells reconstituted with PARP1 variants as indicated 2 days after transfection by immuno-epifluorescence microscopy as shown in Figure 4 (for representative raw data refer to Supplementary Figure S7). Cells were treated with H 2 O 2 for 5 min in concentrations as indicated, and PAR levels of eGFP-positive cells were examined using the anti-PAR-specific mAB 10H. Means ± SEM of n = 5 independent experiments. Statistical analysis was performed using matched two-way ANOVA testing and Sidak's post-test. ( D ) Time-course analysis of PAR formation in PARP1-reconstituted cells after treatment of cells with 250 μM H 2 O 2 . Means ± SEM of n = 4 independent experiments ( > 100 cells per experiment). Statistical analysis using matched two-way ANOVA testing and Sidak's post-test. ( E ) NAD + levels in WT, PARP1 KO and PARP1-reconstituted cells ± H 2 O 2 treatment for 7 min as evaluated by an enzymatic NAD + cycling assay. Means ± SEM of n = 3 independent experiments. Statistical analysis was performed via 2-way ANOVA testing and Sidak's post-test. ( F ) Recruitment and dissociation kinetics of natural PARP1 variants at sites of laser-induced DNA damage. For representative raw data refer to Supplementary Figure S7. Means ± SEM. Evaluation from ≥35 cells from three independent experiments. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test.

    Article Snippet: Orthologous expression and purification of recombinant PARP1 Baculovirus expression constructs of PARP1 were generated according to manufacturer's instructions (BD).

    Techniques: Mutagenesis, Sequencing, Purification, Affinity Chromatography, Activity Assay, Staining, Transfection, Epifluorescence Microscopy

    TALEN-mediated gene targeting of PARP1 in HeLa cells. ( A ) Single-cell immuno-epifluorescence analysis of PARP1 expression in HeLa WT and in two independently generated PARP1 knock-out (KO) clones (KO1 and KO2). ( B ) Western blot analysis of PARP1 expression in HeLa WT and PARP1 KO clones. PCNA served as a loading control. ( C ) Single cell immuno-epifluorescence analysis of PAR formation in HeLa WT and PARP1 KO clones. WT cells showed a dose-dependent increase in cellular PAR levels upon H 2 O 2 treatment (for 5 min), while PAR levels in PARP1 KO cells remained close to background signal intensities. Representative epifluorescent microscopic images (left panel), quantitation of image data (right panel). Means ± SEM, at least 70 cells per data point were analyzed. Statistical analysis was performed via two-way ANOVA testing and Sidak's post-test. ( D) Intracellular NAD + levels in WT and PARP1 KO cells ± H 2 O 2 treatment for 7 min as measured by an enzymatic NAD + cycling assay. Means ± SEM of n = 3 independent experiments. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test. ( E ) Quantitation of basal and H 2 O 2 -induced PAR levels in WT and PARP1 KO cells via isotope dilution mass spectrometry (LC–MS/MS) using a previously published method ( 20 ). To induce PAR-formation, cells were treated with H 2 O 2 as indicated. If indicated, cells were pretreated with 10 μM ABT888 for 45 min. Insert: Basal PAR levels in untreated WT and PARP1 KO cells. Means ± SEM of n = 3 independent experiments. Statistical analysis was performed using one-way ANOVA testing and Sidak's post-test within one group of cells (i.e. WT, KO1, KO2). ( F ) LC–MS/MS analysis of PAR levels ± camptothecin (CPT) treatment for 30 min. Means of n = 2 independent experiments. R-Ado indicates ribosyl-adenosine.

    Journal: Nucleic Acids Research

    Article Title: Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells

    doi: 10.1093/nar/gkw859

    Figure Lengend Snippet: TALEN-mediated gene targeting of PARP1 in HeLa cells. ( A ) Single-cell immuno-epifluorescence analysis of PARP1 expression in HeLa WT and in two independently generated PARP1 knock-out (KO) clones (KO1 and KO2). ( B ) Western blot analysis of PARP1 expression in HeLa WT and PARP1 KO clones. PCNA served as a loading control. ( C ) Single cell immuno-epifluorescence analysis of PAR formation in HeLa WT and PARP1 KO clones. WT cells showed a dose-dependent increase in cellular PAR levels upon H 2 O 2 treatment (for 5 min), while PAR levels in PARP1 KO cells remained close to background signal intensities. Representative epifluorescent microscopic images (left panel), quantitation of image data (right panel). Means ± SEM, at least 70 cells per data point were analyzed. Statistical analysis was performed via two-way ANOVA testing and Sidak's post-test. ( D) Intracellular NAD + levels in WT and PARP1 KO cells ± H 2 O 2 treatment for 7 min as measured by an enzymatic NAD + cycling assay. Means ± SEM of n = 3 independent experiments. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test. ( E ) Quantitation of basal and H 2 O 2 -induced PAR levels in WT and PARP1 KO cells via isotope dilution mass spectrometry (LC–MS/MS) using a previously published method ( 20 ). To induce PAR-formation, cells were treated with H 2 O 2 as indicated. If indicated, cells were pretreated with 10 μM ABT888 for 45 min. Insert: Basal PAR levels in untreated WT and PARP1 KO cells. Means ± SEM of n = 3 independent experiments. Statistical analysis was performed using one-way ANOVA testing and Sidak's post-test within one group of cells (i.e. WT, KO1, KO2). ( F ) LC–MS/MS analysis of PAR levels ± camptothecin (CPT) treatment for 30 min. Means of n = 2 independent experiments. R-Ado indicates ribosyl-adenosine.

    Article Snippet: Orthologous expression and purification of recombinant PARP1 Baculovirus expression constructs of PARP1 were generated according to manufacturer's instructions (BD).

    Techniques: Expressing, Generated, Knock-Out, Western Blot, Clone Assay, Quantitation Assay, Isotope Dilution, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Cycling Probe Technology

    Cellular biochemistry of artificial PARP1 mutants. HeLa PARP1 KO cells were transfected with eGFP-coupled constructs of PARP1, PARP1\L713F and PARP1\E988K. Analyses were performed 2 days after transfection. ( A ) Representative images from single cell immuno-epifluorescence analysis of PARP1-eGFP and PAR after treatment of PARP1-reconstituted cells ± H 2 O 2 as indicated for 5 min. Scale bars indicate 30 μm. ( B ) Densitometric analysis of imaging data as shown in (A). More than 100 cells were analyzed per experiment and condition in a semi-automated manner using a KNIME workflow. Means ± SEM of n = 4 independent experiments. Statistical analysis using matched two-way ANOVA testing and Sidak's post-test. ( C ) Time-course analysis of PAR levels in PARP1-reconstituted cells after treatment of cells with 250 μM H 2 O 2 . Means ± SEM of n = 4 independent experiments, > 100 cells were analyzed per experiment and condition. Statistical analysis was performed using matched two-way ANOVA testing and Sidak's post-test. ( D ) LC–MS/MS analyses of PAR levels in PARP1 KO1 cells and cells reconstituted with PARP1\WT and PARP1\L713F. Two days after transfection, cells were treated as indicated for 7 min. Levels were normalized to transfection efficiencies. Means ± SEM of n = 3 independent experiments. Statistical analysis was performed by one-way ANOVA testing and Tukey's post-test. ( E ) Western blot analysis of HeLa cell extracts of KO1 and PARP1-reconstituted cells, as indicated. 2 days after transfection, cells were treated with 500 μM H 2 O 2 for 7 min. PARylated proteins were detected via the 10H antibody. Red arrows indicate the expected molecular weight of auto-PARylated PARP1. ( F ) NAD + levels in PARP1-reconstituted cells upon treatment ± H 2 O 2 for 7 min as measured by an enzymatic NAD + cycling assay. Means ± SEM of n = 3 independent experiments, except for ABT888-treated samples, n = 2. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test.

    Journal: Nucleic Acids Research

    Article Title: Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells

    doi: 10.1093/nar/gkw859

    Figure Lengend Snippet: Cellular biochemistry of artificial PARP1 mutants. HeLa PARP1 KO cells were transfected with eGFP-coupled constructs of PARP1, PARP1\L713F and PARP1\E988K. Analyses were performed 2 days after transfection. ( A ) Representative images from single cell immuno-epifluorescence analysis of PARP1-eGFP and PAR after treatment of PARP1-reconstituted cells ± H 2 O 2 as indicated for 5 min. Scale bars indicate 30 μm. ( B ) Densitometric analysis of imaging data as shown in (A). More than 100 cells were analyzed per experiment and condition in a semi-automated manner using a KNIME workflow. Means ± SEM of n = 4 independent experiments. Statistical analysis using matched two-way ANOVA testing and Sidak's post-test. ( C ) Time-course analysis of PAR levels in PARP1-reconstituted cells after treatment of cells with 250 μM H 2 O 2 . Means ± SEM of n = 4 independent experiments, > 100 cells were analyzed per experiment and condition. Statistical analysis was performed using matched two-way ANOVA testing and Sidak's post-test. ( D ) LC–MS/MS analyses of PAR levels in PARP1 KO1 cells and cells reconstituted with PARP1\WT and PARP1\L713F. Two days after transfection, cells were treated as indicated for 7 min. Levels were normalized to transfection efficiencies. Means ± SEM of n = 3 independent experiments. Statistical analysis was performed by one-way ANOVA testing and Tukey's post-test. ( E ) Western blot analysis of HeLa cell extracts of KO1 and PARP1-reconstituted cells, as indicated. 2 days after transfection, cells were treated with 500 μM H 2 O 2 for 7 min. PARylated proteins were detected via the 10H antibody. Red arrows indicate the expected molecular weight of auto-PARylated PARP1. ( F ) NAD + levels in PARP1-reconstituted cells upon treatment ± H 2 O 2 for 7 min as measured by an enzymatic NAD + cycling assay. Means ± SEM of n = 3 independent experiments, except for ABT888-treated samples, n = 2. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test.

    Article Snippet: Orthologous expression and purification of recombinant PARP1 Baculovirus expression constructs of PARP1 were generated according to manufacturer's instructions (BD).

    Techniques: Transfection, Construct, Imaging, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Western Blot, Molecular Weight

    Recruitment and dissociation kinetics of PARP1-eGFP at sites of laser-induced DNA damage. ( A ) Representative imaging data. Scale bars indicate 10 μm. ( B ) Densitometric quantitation of signal intensities from imaging data as shown in (A). Means ± SEM of n = 3 independent experiments, > 29 cells were analyzed per experiment and condition. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test.

    Journal: Nucleic Acids Research

    Article Title: Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells

    doi: 10.1093/nar/gkw859

    Figure Lengend Snippet: Recruitment and dissociation kinetics of PARP1-eGFP at sites of laser-induced DNA damage. ( A ) Representative imaging data. Scale bars indicate 10 μm. ( B ) Densitometric quantitation of signal intensities from imaging data as shown in (A). Means ± SEM of n = 3 independent experiments, > 29 cells were analyzed per experiment and condition. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test.

    Article Snippet: Orthologous expression and purification of recombinant PARP1 Baculovirus expression constructs of PARP1 were generated according to manufacturer's instructions (BD).

    Techniques: Imaging, Quantitation Assay

    Overview of PARP1 variants included in this study. ( A ) PARP1 structure and localization of amino acid exchanges of PARP1 variants as used in this study. The 3D structure is in complex with a double-stranded DNA molecule [PDB code 4DQY ( 3 )], without ZnF2 and WGR domains. ( B ) Biochemical parameters derived from rec. enzymes of the different PARP1 variants used in this study. Values were determined in the present study ( cf . Figure 9 ) or taken from the literature as indicated. PAR indicates poly(ADP-ribose); MAR, mono(ADP-ribose); and OAR, oligo(ADP-ribose). ( C ) Western blot analysis of PARP1 protein levels in HeLa PARP1 KO cells reconstituted with different PARP1-eGFP variants 2 days after transfection. Left: representative Western blot out of 4. Right: densitometric analysis of western blot signal intensities after normalization to transfection efficiencies. Means ± SEM of n = 4 independent experiments. Statistical analysis was performed using 1-sample t -test comparing the expression of the different PARP1-variants after transfection to endogenous PARP1\WT-levels in HeLa cells.

    Journal: Nucleic Acids Research

    Article Title: Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells

    doi: 10.1093/nar/gkw859

    Figure Lengend Snippet: Overview of PARP1 variants included in this study. ( A ) PARP1 structure and localization of amino acid exchanges of PARP1 variants as used in this study. The 3D structure is in complex with a double-stranded DNA molecule [PDB code 4DQY ( 3 )], without ZnF2 and WGR domains. ( B ) Biochemical parameters derived from rec. enzymes of the different PARP1 variants used in this study. Values were determined in the present study ( cf . Figure 9 ) or taken from the literature as indicated. PAR indicates poly(ADP-ribose); MAR, mono(ADP-ribose); and OAR, oligo(ADP-ribose). ( C ) Western blot analysis of PARP1 protein levels in HeLa PARP1 KO cells reconstituted with different PARP1-eGFP variants 2 days after transfection. Left: representative Western blot out of 4. Right: densitometric analysis of western blot signal intensities after normalization to transfection efficiencies. Means ± SEM of n = 4 independent experiments. Statistical analysis was performed using 1-sample t -test comparing the expression of the different PARP1-variants after transfection to endogenous PARP1\WT-levels in HeLa cells.

    Article Snippet: Orthologous expression and purification of recombinant PARP1 Baculovirus expression constructs of PARP1 were generated according to manufacturer's instructions (BD).

    Techniques: Derivative Assay, Western Blot, Transfection, Expressing

    Functional consequences of PARP1 deletion in HeLa cells. ( A ) Cell proliferation of HeLa WT and PARP1 KO cells as analyzed by Alamar Blue assay for 3 days. Means ± SEM of n = 3 independent experiments. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test. ( B ) Cell cycle analysis of untreated WT and PARP1 KO cells via PI staining and subsequent flow cytometric analysis. Means ± SEM of three independent experiments. ( C ) Clonogenic survival analysis. HeLa WT and PARP1 KO cells were treated with H 2 O 2 as indicated for 5 min and then plated and cultivated for 2 weeks prior to colony counting. Means ± SEM of n = 3 independent experiments. Statistical analysis using two-way ANOVA testing and Sidak's post-test. ( D ) Cytotoxicity analysis via annexin V/PI staining and subsequent flow cytometric analysis of HeLa WT and PARP1 KO cells treated ± CPT in concentrations as indicated for 2 days. Viable cells refer to annexin V/PI-double negative cells (top); early apoptotic cells to annexin V-positive (middle), PI-negative cells; and necrotic and late-apoptotic cells to annexin V/PI-double positive cells (bottom). Ratios compared to total cell numbers. Means ± SEM of n ≥ 4 independent experiments. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test. ( E ) Cell cycle analysis via PI staining and flow cytometric analysis 2 days after treatment of cells ± CPT in concentrations as indicated. Means ± SEM of n ≥ 4 independent experiments except for data of PARP1 KO2 cells; n = 1. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test.

    Journal: Nucleic Acids Research

    Article Title: Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells

    doi: 10.1093/nar/gkw859

    Figure Lengend Snippet: Functional consequences of PARP1 deletion in HeLa cells. ( A ) Cell proliferation of HeLa WT and PARP1 KO cells as analyzed by Alamar Blue assay for 3 days. Means ± SEM of n = 3 independent experiments. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test. ( B ) Cell cycle analysis of untreated WT and PARP1 KO cells via PI staining and subsequent flow cytometric analysis. Means ± SEM of three independent experiments. ( C ) Clonogenic survival analysis. HeLa WT and PARP1 KO cells were treated with H 2 O 2 as indicated for 5 min and then plated and cultivated for 2 weeks prior to colony counting. Means ± SEM of n = 3 independent experiments. Statistical analysis using two-way ANOVA testing and Sidak's post-test. ( D ) Cytotoxicity analysis via annexin V/PI staining and subsequent flow cytometric analysis of HeLa WT and PARP1 KO cells treated ± CPT in concentrations as indicated for 2 days. Viable cells refer to annexin V/PI-double negative cells (top); early apoptotic cells to annexin V-positive (middle), PI-negative cells; and necrotic and late-apoptotic cells to annexin V/PI-double positive cells (bottom). Ratios compared to total cell numbers. Means ± SEM of n ≥ 4 independent experiments. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test. ( E ) Cell cycle analysis via PI staining and flow cytometric analysis 2 days after treatment of cells ± CPT in concentrations as indicated. Means ± SEM of n ≥ 4 independent experiments except for data of PARP1 KO2 cells; n = 1. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test.

    Article Snippet: Orthologous expression and purification of recombinant PARP1 Baculovirus expression constructs of PARP1 were generated according to manufacturer's instructions (BD).

    Techniques: Functional Assay, Alamar Blue Assay, Cell Cycle Assay, Staining, Flow Cytometry, Cycling Probe Technology

    PARP1\E988K affects cell cycle regulation and induces DNA damage signaling. ( A ) Cell cycle analysis by PI staining and subsequent flow cytometric analysis 3 days after transfection of HeLa PARP1 KO cells reconstituted with PARP1\WT, PARP1\E988K and PARP1\L713F. PARP1\E988K induces a G2 arrest, which can be rescued by treating cells with 10 μM ABT888. Means ± SEM of n = 6 independent experiments, except of ABT888-treated samples, n = 2. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test. ( B ) Analysis of DNA damage response markers, i.e. phospho-S15-p53, γH2A.X and p16 in WT, PARP1 KO and PARP1-reconstituted cells as indicated 2 days after transfection. Cells reconstituted with PARP1\E988K displayed increased phospho-S15-p53 and γH2A.X levels and cells reconstituted with PARP1\L713F showed slightly elevated γH2A.X levels. Immunochemical detection of PARP1, p53 and actin served as controls. Shown is a representative experiment out of three. ( C ) Western blot analysis of ph-p53 and γH2A.X levels in PARP1-reconstituted cells (±10-μM ABT888 treatment). ( D ) Western blot analysis of the replicative stress marker phospho-RPA2 (Ser4/8) in PARP1-reconstituted cells (±10-μM ABT888 treatment). Immunochemical detection of PARP1 and actin served as controls.

    Journal: Nucleic Acids Research

    Article Title: Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells

    doi: 10.1093/nar/gkw859

    Figure Lengend Snippet: PARP1\E988K affects cell cycle regulation and induces DNA damage signaling. ( A ) Cell cycle analysis by PI staining and subsequent flow cytometric analysis 3 days after transfection of HeLa PARP1 KO cells reconstituted with PARP1\WT, PARP1\E988K and PARP1\L713F. PARP1\E988K induces a G2 arrest, which can be rescued by treating cells with 10 μM ABT888. Means ± SEM of n = 6 independent experiments, except of ABT888-treated samples, n = 2. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test. ( B ) Analysis of DNA damage response markers, i.e. phospho-S15-p53, γH2A.X and p16 in WT, PARP1 KO and PARP1-reconstituted cells as indicated 2 days after transfection. Cells reconstituted with PARP1\E988K displayed increased phospho-S15-p53 and γH2A.X levels and cells reconstituted with PARP1\L713F showed slightly elevated γH2A.X levels. Immunochemical detection of PARP1, p53 and actin served as controls. Shown is a representative experiment out of three. ( C ) Western blot analysis of ph-p53 and γH2A.X levels in PARP1-reconstituted cells (±10-μM ABT888 treatment). ( D ) Western blot analysis of the replicative stress marker phospho-RPA2 (Ser4/8) in PARP1-reconstituted cells (±10-μM ABT888 treatment). Immunochemical detection of PARP1 and actin served as controls.

    Article Snippet: Orthologous expression and purification of recombinant PARP1 Baculovirus expression constructs of PARP1 were generated according to manufacturer's instructions (BD).

    Techniques: Cell Cycle Assay, Staining, Flow Cytometry, Transfection, Western Blot, Marker

    PARP1 mutants influence cell viability and cell cycle progression upon CPT treatment. ( A and B ) Analysis of viable, apoptotic and necrotic cells 3 days after transfection of HeLa PARP1 KO cells reconstituted with PARP1, PARP1\E988K, and PARP1\L713F by annexinV / PI staining and subsequent flow cytometric analysis. GFP cont indicates cells transfected with a plasmid carrying only GFP. Cells were treated ( A ) with CPT in concentrations as indicated 24 h after transfection or ( B ) with 10 μM ABT888 directly after transfection. Viable cells refer to annexin V/PI-double negative cells; (early) apoptotic cells to annexin V-positive, PI-negative cells; and necrotic and late-apoptotic cells to annexin V/PI-double positive cells (ratios compared to total cell numbers). Means ± SEM of n ≥ 3 independent experiments. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test. ( C ) Cell cycle analysis of PARP1-reconstituted cells as indicated 3 days after transfection and 2 days after CPT treatment via PI staining and subsequent flow cytometric analysis. Means ± SEM of n = 3 independent experiments. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test.

    Journal: Nucleic Acids Research

    Article Title: Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells

    doi: 10.1093/nar/gkw859

    Figure Lengend Snippet: PARP1 mutants influence cell viability and cell cycle progression upon CPT treatment. ( A and B ) Analysis of viable, apoptotic and necrotic cells 3 days after transfection of HeLa PARP1 KO cells reconstituted with PARP1, PARP1\E988K, and PARP1\L713F by annexinV / PI staining and subsequent flow cytometric analysis. GFP cont indicates cells transfected with a plasmid carrying only GFP. Cells were treated ( A ) with CPT in concentrations as indicated 24 h after transfection or ( B ) with 10 μM ABT888 directly after transfection. Viable cells refer to annexin V/PI-double negative cells; (early) apoptotic cells to annexin V-positive, PI-negative cells; and necrotic and late-apoptotic cells to annexin V/PI-double positive cells (ratios compared to total cell numbers). Means ± SEM of n ≥ 3 independent experiments. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test. ( C ) Cell cycle analysis of PARP1-reconstituted cells as indicated 3 days after transfection and 2 days after CPT treatment via PI staining and subsequent flow cytometric analysis. Means ± SEM of n = 3 independent experiments. Statistical analysis was performed using two-way ANOVA testing and Sidak's post-test.

    Article Snippet: Orthologous expression and purification of recombinant PARP1 Baculovirus expression constructs of PARP1 were generated according to manufacturer's instructions (BD).

    Techniques: Cycling Probe Technology, Transfection, Staining, Flow Cytometry, Plasmid Preparation, Cell Cycle Assay

    Non-covalent PARP1–PAR interaction. ( A ) Analysis of PARP1–PAR interaction by PAR overlay assay using increasing amounts of recombinant PARP1 as indicated. After protein transfer, membranes were incubated with (left panel) or without (right panel) purified PAR (0.2 μM). PAR binding was detected using the 10H antibody after high-stringency washing to remove non-specifically bound PAR. ( B ) Immuno slot-blot PAR binding assay using increasing amounts of recombinant PARP1. Membranes were incubated with PAR (0.2 μM) and bound PAR was detected using the 10H antibody after high-stringency washing. ( C ) Analysis of PARP1–PAR interaction in solution using a modified PAR-EMSA. End-biotinylated PAR of defined chain length (30–35 mer, 0.5 pmol ADP-ribose) was incubated with recombinant PARP1. PARP1–PAR binding was assessed by native TBE gel electrophoreses and Western blotting. Three distinct complexes (1–3) were formed in a PARP1-dependent manner. ( D ) Upper panel. In silico search for putative PAR-binding sites within the PARP1 sequence using the search sequence displayed at the top of the panel. Two potential PAR binding motives (PBMs), i.e. PBM1 (1 mismatch) and PBM2 (two mismatches), were identified in Zn2 and Zn3, respectively. Lower panels . Localization of PBM1 and PBM2 within Zn2 and Zn3, respectively. Structures based on PDB codes 4AV1 and 4DQY ( 3 ). ( E ) PAR binding ability of peptides comprising aa sequences of PBM1/2 and peptides comprising aa exchanges potentially responsible of PBM-PAR interactions using a PepSpot analysis. ‘AA pos.’ indicates aa positions within full-length PARP1 sequence ( 85 ). A peptide sequence derived from a PBM in XRCC1 served as a positive control. ( F ) DNA-PARP1 EMSA using a known biotinylated double-stranded DNA oligonucleotide (200 fmol). Left . DNA-PARP1 interaction in the absence of PAR. Middle . Rec. PARP1 (100 nM) was incubated with increasing concentrations of PAR as indicated. Right . Densitometric evaluation of EMSAs. Means ± SEM of n = 3 independent experiments. Statistical analysis was performed using 1-way ANOVA testing and Sidak's post-test.

    Journal: Nucleic Acids Research

    Article Title: Analyzing structure–function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells

    doi: 10.1093/nar/gkw859

    Figure Lengend Snippet: Non-covalent PARP1–PAR interaction. ( A ) Analysis of PARP1–PAR interaction by PAR overlay assay using increasing amounts of recombinant PARP1 as indicated. After protein transfer, membranes were incubated with (left panel) or without (right panel) purified PAR (0.2 μM). PAR binding was detected using the 10H antibody after high-stringency washing to remove non-specifically bound PAR. ( B ) Immuno slot-blot PAR binding assay using increasing amounts of recombinant PARP1. Membranes were incubated with PAR (0.2 μM) and bound PAR was detected using the 10H antibody after high-stringency washing. ( C ) Analysis of PARP1–PAR interaction in solution using a modified PAR-EMSA. End-biotinylated PAR of defined chain length (30–35 mer, 0.5 pmol ADP-ribose) was incubated with recombinant PARP1. PARP1–PAR binding was assessed by native TBE gel electrophoreses and Western blotting. Three distinct complexes (1–3) were formed in a PARP1-dependent manner. ( D ) Upper panel. In silico search for putative PAR-binding sites within the PARP1 sequence using the search sequence displayed at the top of the panel. Two potential PAR binding motives (PBMs), i.e. PBM1 (1 mismatch) and PBM2 (two mismatches), were identified in Zn2 and Zn3, respectively. Lower panels . Localization of PBM1 and PBM2 within Zn2 and Zn3, respectively. Structures based on PDB codes 4AV1 and 4DQY ( 3 ). ( E ) PAR binding ability of peptides comprising aa sequences of PBM1/2 and peptides comprising aa exchanges potentially responsible of PBM-PAR interactions using a PepSpot analysis. ‘AA pos.’ indicates aa positions within full-length PARP1 sequence ( 85 ). A peptide sequence derived from a PBM in XRCC1 served as a positive control. ( F ) DNA-PARP1 EMSA using a known biotinylated double-stranded DNA oligonucleotide (200 fmol). Left . DNA-PARP1 interaction in the absence of PAR. Middle . Rec. PARP1 (100 nM) was incubated with increasing concentrations of PAR as indicated. Right . Densitometric evaluation of EMSAs. Means ± SEM of n = 3 independent experiments. Statistical analysis was performed using 1-way ANOVA testing and Sidak's post-test.

    Article Snippet: Orthologous expression and purification of recombinant PARP1 Baculovirus expression constructs of PARP1 were generated according to manufacturer's instructions (BD).

    Techniques: Overlay Assay, Recombinant, Incubation, Purification, Binding Assay, Dot Blot, Modification, Western Blot, In Silico, Sequencing, Derivative Assay, Positive Control

    JNK2, but not JNK1, is essential for virus-induced apoptosis. ( A ) Control, JNK1 and JNK2 siRNA knock-down HEK293 cells ( left ), or wild type, Jnk1 −/− and Jnk2 −/− MEF cells ( right ), were treated with SeV (for HEK293 MOI = 1, for MEF MOI = 4) for the indicated times. Cell lysates were analyzed by western blot, probing for ISG15, ISG60, JNK1 and JNK2 with the indicated antibodies. ( B ) Wild type and Mavs −/− MEF cells were treated with SeV (MOI = 4), or TNF-α (10 ng/ml) plus cycloheximide (CHX, 10 µg/ml) for the indicated times. Cell lysates were collected for western blot analysis using anti-PARP antibody to determine cell apoptosis and using anti-MAVS antibody to measure the deficiency of MAVS protein. ( C ) HEK293 cells were transfected with the indicated plasmids and 24 hours later, cell lysates were collected for western blot analysis of PARP, phosphorylated JNK, phosphorylated IRF3, Flag-tagged proteins and β-actin. ( D ) HEK293 cells were treated by SeV (MOI = 1) with or without JNK kinase inhibitor SP600125 (5 µM). Cell lysates were collected for western blot analysis of PARP, cleaved caspase-3 and β-actin to probe for cell apoptosis. ( E ) Control, JNK1 or JNK2 knocked down HEK293 cells were treated with SeV (MOI = 1) for the indicated times. Cell lysates were collected for western blot analysis to measure cell apoptosis using the indicated antibodies. ( F ) Wild type, Jnk1 −/− or Jnk2 −/− MEF cells were treated with SeV (MOI = 4) for the indicated times. Cell lysates were collected for western blot analysis.

    Journal: PLoS Pathogens

    Article Title: MAVS-MKK7-JNK2 Defines a Novel Apoptotic Signaling Pathway during Viral Infection

    doi: 10.1371/journal.ppat.1004020

    Figure Lengend Snippet: JNK2, but not JNK1, is essential for virus-induced apoptosis. ( A ) Control, JNK1 and JNK2 siRNA knock-down HEK293 cells ( left ), or wild type, Jnk1 −/− and Jnk2 −/− MEF cells ( right ), were treated with SeV (for HEK293 MOI = 1, for MEF MOI = 4) for the indicated times. Cell lysates were analyzed by western blot, probing for ISG15, ISG60, JNK1 and JNK2 with the indicated antibodies. ( B ) Wild type and Mavs −/− MEF cells were treated with SeV (MOI = 4), or TNF-α (10 ng/ml) plus cycloheximide (CHX, 10 µg/ml) for the indicated times. Cell lysates were collected for western blot analysis using anti-PARP antibody to determine cell apoptosis and using anti-MAVS antibody to measure the deficiency of MAVS protein. ( C ) HEK293 cells were transfected with the indicated plasmids and 24 hours later, cell lysates were collected for western blot analysis of PARP, phosphorylated JNK, phosphorylated IRF3, Flag-tagged proteins and β-actin. ( D ) HEK293 cells were treated by SeV (MOI = 1) with or without JNK kinase inhibitor SP600125 (5 µM). Cell lysates were collected for western blot analysis of PARP, cleaved caspase-3 and β-actin to probe for cell apoptosis. ( E ) Control, JNK1 or JNK2 knocked down HEK293 cells were treated with SeV (MOI = 1) for the indicated times. Cell lysates were collected for western blot analysis to measure cell apoptosis using the indicated antibodies. ( F ) Wild type, Jnk1 −/− or Jnk2 −/− MEF cells were treated with SeV (MOI = 4) for the indicated times. Cell lysates were collected for western blot analysis.

    Article Snippet: The following antibodies were used for western blot or immunoprecipitation: anti-β-actin (A5316, Sigma), normal mouse IgG (sc-2025, Santa Cruz Biotechnology), normal rabbit IgG (sc-2027, Santa Cruz Biotechnology), anti-HA (sc-7392, Santa Cruz Biotechnology), anti-Flag (F1804, Sigma), anti-Tom20 (11802-1-AP, Proteintech; sc-17764, Santa Cruz Biotechnology), anti-Caspase-3 (9662, Cell Signaling; 9661, Cell Signaling), anti-PARP (sc-7150, Santa Cruz Biotechnology; 9542, Cell Signaling), anti-MAVS (generated by this laboratory and also purchased from Cell Signaling–3993), anti-JNK (sc-571, Santa Cruz Biotechnology), anti-p-JNK (9255, Cell Signaling), anti-p38 (sc-7149, Santa Cruz Biotechnology), anti-p-p38 (9211, Cell Signaling), anti-ERK (9102, Cell Signaling), anti-p-ERK (9101, Cell Signaling), anti-TBK1 (sc-73115, Santa Cruz Biotechnology), anti-ISG15 (M24004, Abmart), anti-ISG60 (15201-1-AP, Proteintech), anti-MKK4 (sc964, Santa Cruz Biotechnology), anti-MKK7 (generated by this laboratory and also purchased from Abcam–ab52618), anti-p-IRF3 (4947, Cell Signaling), anti-TRAF2 (sc-876, Santa Cruz Biotechnology), anti-TRAF3 (sc-1828, Santa Cruz Biotechnology), anti-TRADD (sc-46653, Santa Cruz Biotechnology), anti-RIG-I (AB54008, Shanghai Sangon Biotech; 4520, Cell Signaling), anti-MDA5 (5321, Cell Signaling), anti-MKK3 (5674, Cell Signaling) and anti-MKK6 (9264, Cell Signaling).

    Techniques: Western Blot, Transfection

    MKK7 functionally links MAVS to JNK2 during virus-induced apoptosis. ( A ) Wild type, Mkk3/6 −/− or Mkk4/7 −/− MEF cells were treated with SeV (MOI = 4) for the indicated times. Cell lysates were collected for western blot analysis using anti-JNK, anti-p-JNK, anti-MKK3, anti-MKK4, anti-MKK6, anti-MKK7 and anti-β-actin antibodies. ( B ) HEK293 cells were transfected with NC, MKK4 and MKK7 siRNAs respectively for 48 hours and then treated by SeV (MOI = 1) for the indicated times. Cell lysates were collected for western blot analysis of p-JNK, JNK, MKK4, MKK7 and β-actin. ( C ) Wild type, Mkk3/6 −/− or Mkk4/7 −/− MEF cells were treated with SeV (MOI = 4) for the indicated times. Cell apoptosis was determined by western blot analysis of activated PARP and cleaved caspase-3. ( D ) HEK293 cells were transfected with NC, MKK4 and MKK7 siRNAs for 48 hours and then treated with SeV (MOI = 1) for 24 hours. Cell lysates were collected for western blot analysis of PARP and cleaved caspase-3 to measure cell apoptosis. Mkk3/6 −/− , Mkk3 and Mkk6 double knockout; Mkk4/7 −/− , Mkk4 and Mkk7 double knockout.

    Journal: PLoS Pathogens

    Article Title: MAVS-MKK7-JNK2 Defines a Novel Apoptotic Signaling Pathway during Viral Infection

    doi: 10.1371/journal.ppat.1004020

    Figure Lengend Snippet: MKK7 functionally links MAVS to JNK2 during virus-induced apoptosis. ( A ) Wild type, Mkk3/6 −/− or Mkk4/7 −/− MEF cells were treated with SeV (MOI = 4) for the indicated times. Cell lysates were collected for western blot analysis using anti-JNK, anti-p-JNK, anti-MKK3, anti-MKK4, anti-MKK6, anti-MKK7 and anti-β-actin antibodies. ( B ) HEK293 cells were transfected with NC, MKK4 and MKK7 siRNAs respectively for 48 hours and then treated by SeV (MOI = 1) for the indicated times. Cell lysates were collected for western blot analysis of p-JNK, JNK, MKK4, MKK7 and β-actin. ( C ) Wild type, Mkk3/6 −/− or Mkk4/7 −/− MEF cells were treated with SeV (MOI = 4) for the indicated times. Cell apoptosis was determined by western blot analysis of activated PARP and cleaved caspase-3. ( D ) HEK293 cells were transfected with NC, MKK4 and MKK7 siRNAs for 48 hours and then treated with SeV (MOI = 1) for 24 hours. Cell lysates were collected for western blot analysis of PARP and cleaved caspase-3 to measure cell apoptosis. Mkk3/6 −/− , Mkk3 and Mkk6 double knockout; Mkk4/7 −/− , Mkk4 and Mkk7 double knockout.

    Article Snippet: The following antibodies were used for western blot or immunoprecipitation: anti-β-actin (A5316, Sigma), normal mouse IgG (sc-2025, Santa Cruz Biotechnology), normal rabbit IgG (sc-2027, Santa Cruz Biotechnology), anti-HA (sc-7392, Santa Cruz Biotechnology), anti-Flag (F1804, Sigma), anti-Tom20 (11802-1-AP, Proteintech; sc-17764, Santa Cruz Biotechnology), anti-Caspase-3 (9662, Cell Signaling; 9661, Cell Signaling), anti-PARP (sc-7150, Santa Cruz Biotechnology; 9542, Cell Signaling), anti-MAVS (generated by this laboratory and also purchased from Cell Signaling–3993), anti-JNK (sc-571, Santa Cruz Biotechnology), anti-p-JNK (9255, Cell Signaling), anti-p38 (sc-7149, Santa Cruz Biotechnology), anti-p-p38 (9211, Cell Signaling), anti-ERK (9102, Cell Signaling), anti-p-ERK (9101, Cell Signaling), anti-TBK1 (sc-73115, Santa Cruz Biotechnology), anti-ISG15 (M24004, Abmart), anti-ISG60 (15201-1-AP, Proteintech), anti-MKK4 (sc964, Santa Cruz Biotechnology), anti-MKK7 (generated by this laboratory and also purchased from Abcam–ab52618), anti-p-IRF3 (4947, Cell Signaling), anti-TRAF2 (sc-876, Santa Cruz Biotechnology), anti-TRAF3 (sc-1828, Santa Cruz Biotechnology), anti-TRADD (sc-46653, Santa Cruz Biotechnology), anti-RIG-I (AB54008, Shanghai Sangon Biotech; 4520, Cell Signaling), anti-MDA5 (5321, Cell Signaling), anti-MKK3 (5674, Cell Signaling) and anti-MKK6 (9264, Cell Signaling).

    Techniques: Western Blot, Transfection, Double Knockout

    MAVS-MKK7-JNK2 represents a novel apoptotic signaling cascade. ( A ) Wild type and Mkk4/7 −/− MEF cells were transfected with the indicated combinations of plasmids for 24 hours. Mkk4/7 −/− MEFs with HA-MKK4 ectopic-expression were used to mimic Mkk7 −/− and Mkk4/7 −/− MEFs with HA-MKK7 ectopic-expression were used to mimic Mkk4 −/− . Cell lysates were collected for western blot analysis using the indicated antibodies. ( B ) Wild type, Jnk1 −/− or Jnk2 −/− MEF cells were transfected with the plasmid Flag-MAVS at a dose gradient (0, 2 and 4 µg/well in a 12-well plate). Cell lysates were collected for western blot analysis using the indicated antibodies. ( C ) Wild type or truncation mutants of MKK7 were re-introduced into “ Mkk7 −/− MEF cells” for 24 hours and the cells were then infected with SeV for 48 hours. Cell apoptosis was measured by western blot analysis of PARP and cleaved caspase-3. ( D ) HA-tagged JNK1, JNK2 and mutated JNK2(183/185A) were re-introduced into Jnk2 −/− MEF cells, followed by similar analysis as described in C . ( E ) Flag-tagged wild type MAVS and truncation mutant MAVS-ΔTM were re-introduced into Mavs −/− MEF cells, followed by similar analysis as described in C .

    Journal: PLoS Pathogens

    Article Title: MAVS-MKK7-JNK2 Defines a Novel Apoptotic Signaling Pathway during Viral Infection

    doi: 10.1371/journal.ppat.1004020

    Figure Lengend Snippet: MAVS-MKK7-JNK2 represents a novel apoptotic signaling cascade. ( A ) Wild type and Mkk4/7 −/− MEF cells were transfected with the indicated combinations of plasmids for 24 hours. Mkk4/7 −/− MEFs with HA-MKK4 ectopic-expression were used to mimic Mkk7 −/− and Mkk4/7 −/− MEFs with HA-MKK7 ectopic-expression were used to mimic Mkk4 −/− . Cell lysates were collected for western blot analysis using the indicated antibodies. ( B ) Wild type, Jnk1 −/− or Jnk2 −/− MEF cells were transfected with the plasmid Flag-MAVS at a dose gradient (0, 2 and 4 µg/well in a 12-well plate). Cell lysates were collected for western blot analysis using the indicated antibodies. ( C ) Wild type or truncation mutants of MKK7 were re-introduced into “ Mkk7 −/− MEF cells” for 24 hours and the cells were then infected with SeV for 48 hours. Cell apoptosis was measured by western blot analysis of PARP and cleaved caspase-3. ( D ) HA-tagged JNK1, JNK2 and mutated JNK2(183/185A) were re-introduced into Jnk2 −/− MEF cells, followed by similar analysis as described in C . ( E ) Flag-tagged wild type MAVS and truncation mutant MAVS-ΔTM were re-introduced into Mavs −/− MEF cells, followed by similar analysis as described in C .

    Article Snippet: The following antibodies were used for western blot or immunoprecipitation: anti-β-actin (A5316, Sigma), normal mouse IgG (sc-2025, Santa Cruz Biotechnology), normal rabbit IgG (sc-2027, Santa Cruz Biotechnology), anti-HA (sc-7392, Santa Cruz Biotechnology), anti-Flag (F1804, Sigma), anti-Tom20 (11802-1-AP, Proteintech; sc-17764, Santa Cruz Biotechnology), anti-Caspase-3 (9662, Cell Signaling; 9661, Cell Signaling), anti-PARP (sc-7150, Santa Cruz Biotechnology; 9542, Cell Signaling), anti-MAVS (generated by this laboratory and also purchased from Cell Signaling–3993), anti-JNK (sc-571, Santa Cruz Biotechnology), anti-p-JNK (9255, Cell Signaling), anti-p38 (sc-7149, Santa Cruz Biotechnology), anti-p-p38 (9211, Cell Signaling), anti-ERK (9102, Cell Signaling), anti-p-ERK (9101, Cell Signaling), anti-TBK1 (sc-73115, Santa Cruz Biotechnology), anti-ISG15 (M24004, Abmart), anti-ISG60 (15201-1-AP, Proteintech), anti-MKK4 (sc964, Santa Cruz Biotechnology), anti-MKK7 (generated by this laboratory and also purchased from Abcam–ab52618), anti-p-IRF3 (4947, Cell Signaling), anti-TRAF2 (sc-876, Santa Cruz Biotechnology), anti-TRAF3 (sc-1828, Santa Cruz Biotechnology), anti-TRADD (sc-46653, Santa Cruz Biotechnology), anti-RIG-I (AB54008, Shanghai Sangon Biotech; 4520, Cell Signaling), anti-MDA5 (5321, Cell Signaling), anti-MKK3 (5674, Cell Signaling) and anti-MKK6 (9264, Cell Signaling).

    Techniques: Transfection, Expressing, Western Blot, Plasmid Preparation, Infection, Mutagenesis

    Effect of poly(ADP-ribosyl)ated PARP-1 on DNA joining by DNA ligase III. (A) Purified PARP-1 (100 nM) was preincubated with a labeled DNA duplex containing a single ligatable nick (50 nM) in the presence (+) or absence (−) of NAD. Subsequently, intact DNA ligase III (Lig III; 5 nM) or a truncated version lacking the zinc finger (ΔZf-Lig III; 5 nM) was added to the reaction mixture as indicated. After incubation for 10 min at 25°C, labeled oligonucleotides were separated by denaturing gel electrophoresis. The labeled substrate (30-mer) and ligated product (50-mer) were detected and quantitated by phosphorimager analysis. (B) The results of three independent DNA joining assays are shown graphically. White bars, full-length DNA ligase IIIβ; grey bars, truncated version of DNA ligase IIIβ lacking the zinc finger.

    Journal: Molecular and Cellular Biology

    Article Title: Physical and Functional Interaction between DNA Ligase III? and Poly(ADP-Ribose) Polymerase 1 in DNA Single-Strand Break Repair

    doi: 10.1128/MCB.23.16.5919-5927.2003

    Figure Lengend Snippet: Effect of poly(ADP-ribosyl)ated PARP-1 on DNA joining by DNA ligase III. (A) Purified PARP-1 (100 nM) was preincubated with a labeled DNA duplex containing a single ligatable nick (50 nM) in the presence (+) or absence (−) of NAD. Subsequently, intact DNA ligase III (Lig III; 5 nM) or a truncated version lacking the zinc finger (ΔZf-Lig III; 5 nM) was added to the reaction mixture as indicated. After incubation for 10 min at 25°C, labeled oligonucleotides were separated by denaturing gel electrophoresis. The labeled substrate (30-mer) and ligated product (50-mer) were detected and quantitated by phosphorimager analysis. (B) The results of three independent DNA joining assays are shown graphically. White bars, full-length DNA ligase IIIβ; grey bars, truncated version of DNA ligase IIIβ lacking the zinc finger.

    Article Snippet: Based on its relative abundance and high binding affinity, it seems likely that PARP-1 is the first factor to bind to DNA single-strand breaks in vivo (Fig. ), resulting in activation of its polymerase activity and automodification since PARP-1 itself is the major acceptor for PAR ( ).

    Techniques: Purification, Labeling, Incubation, Nucleic Acid Electrophoresis

    (A) The yeast two-hybrid genetic screen was employed to determine whether DNA ligase III interacts directly with PARP-1. Yeast strains were constructed as described in Materials and Methods. Colonies were tested for growth on triple dropout medium (-Leu,-Trp,-His) and subjected to a β-galactosidase (β-Gal) filter assay to detect protein-protein interactions as indicated. P/LIII, yeast strain expressing PARP-1 fused to the GAL4 activation domain (P) and DNA ligase III fused to the GAL4 DNA binding domain (LIII); P/V, yeast strain expressing the GAL4 binding domain (V) and PARP-1 fused to the GAL4 activation domain (P). (B) Mapping of the region of DNA ligase III that interacts with PARP-1. Pull-down assays with glutathione Sepharose and nickel beads were performed as described in Materials and Methods with purified PARP-1 (10 nM) and tagged versions of DNA ligase III. Ni, nickel beads only; Ni-Lig III, nickel beads liganded by His-tagged full-length DNA ligase III; Ni-ΔZf-LIII, nickel beads liganded by His-tagged DNA ligase III lacking the N-terminal 55 amino acids; GST, glutathione Sepharose beads liganded by GST; GST-692, glutathione Sepharose beads liganded by a GST fusion protein containing the C-terminal 692 residues of DNA ligase III; GST-152, glutathione Sepharose beads liganded by a GST fusion protein containing the N-terminal 152 residues of DNA ligase III; Zf, zinc finger; cat. domain, catalytic domain. The binding of PARP-1 to the beads was detected by immunoblotting (IB) with PARP-1 monoclonal antibody.

    Journal: Molecular and Cellular Biology

    Article Title: Physical and Functional Interaction between DNA Ligase III? and Poly(ADP-Ribose) Polymerase 1 in DNA Single-Strand Break Repair

    doi: 10.1128/MCB.23.16.5919-5927.2003

    Figure Lengend Snippet: (A) The yeast two-hybrid genetic screen was employed to determine whether DNA ligase III interacts directly with PARP-1. Yeast strains were constructed as described in Materials and Methods. Colonies were tested for growth on triple dropout medium (-Leu,-Trp,-His) and subjected to a β-galactosidase (β-Gal) filter assay to detect protein-protein interactions as indicated. P/LIII, yeast strain expressing PARP-1 fused to the GAL4 activation domain (P) and DNA ligase III fused to the GAL4 DNA binding domain (LIII); P/V, yeast strain expressing the GAL4 binding domain (V) and PARP-1 fused to the GAL4 activation domain (P). (B) Mapping of the region of DNA ligase III that interacts with PARP-1. Pull-down assays with glutathione Sepharose and nickel beads were performed as described in Materials and Methods with purified PARP-1 (10 nM) and tagged versions of DNA ligase III. Ni, nickel beads only; Ni-Lig III, nickel beads liganded by His-tagged full-length DNA ligase III; Ni-ΔZf-LIII, nickel beads liganded by His-tagged DNA ligase III lacking the N-terminal 55 amino acids; GST, glutathione Sepharose beads liganded by GST; GST-692, glutathione Sepharose beads liganded by a GST fusion protein containing the C-terminal 692 residues of DNA ligase III; GST-152, glutathione Sepharose beads liganded by a GST fusion protein containing the N-terminal 152 residues of DNA ligase III; Zf, zinc finger; cat. domain, catalytic domain. The binding of PARP-1 to the beads was detected by immunoblotting (IB) with PARP-1 monoclonal antibody.

    Article Snippet: Based on its relative abundance and high binding affinity, it seems likely that PARP-1 is the first factor to bind to DNA single-strand breaks in vivo (Fig. ), resulting in activation of its polymerase activity and automodification since PARP-1 itself is the major acceptor for PAR ( ).

    Techniques: Construct, Expressing, Activation Assay, Binding Assay, Purification

    Model of the repair of DNA single-strand breaks by PARP-1 and DNA ligase III-XRCC1. The binding of PARP-1 to a DNA single-strand break activates PARP-1's polymerase activity that in turn results in automodification and dissociation of poly(ADP-ribosyl)ated PARP-1 from the strand break. DNA ligase IIIα (LigIII)-XRCC1 (XR1) associates with poly(ADP-ribosyl)ated PARP-1 in the vicinity of the DNA strand break. The zinc finger of DNA ligase IIIα enables this ternary complex to specifically recognize and bind to the DNA strand break despite the presence of negatively charged PARs. Additional repair factors such as polynucleotide kinase (PNK), Pol β, and AP endonuclease (APE) that process damaged termini are recruited via interactions with XRCC1. After the generation of ligatable termini, repair is completed by DNA ligase IIIα.

    Journal: Molecular and Cellular Biology

    Article Title: Physical and Functional Interaction between DNA Ligase III? and Poly(ADP-Ribose) Polymerase 1 in DNA Single-Strand Break Repair

    doi: 10.1128/MCB.23.16.5919-5927.2003

    Figure Lengend Snippet: Model of the repair of DNA single-strand breaks by PARP-1 and DNA ligase III-XRCC1. The binding of PARP-1 to a DNA single-strand break activates PARP-1's polymerase activity that in turn results in automodification and dissociation of poly(ADP-ribosyl)ated PARP-1 from the strand break. DNA ligase IIIα (LigIII)-XRCC1 (XR1) associates with poly(ADP-ribosyl)ated PARP-1 in the vicinity of the DNA strand break. The zinc finger of DNA ligase IIIα enables this ternary complex to specifically recognize and bind to the DNA strand break despite the presence of negatively charged PARs. Additional repair factors such as polynucleotide kinase (PNK), Pol β, and AP endonuclease (APE) that process damaged termini are recruited via interactions with XRCC1. After the generation of ligatable termini, repair is completed by DNA ligase IIIα.

    Article Snippet: Based on its relative abundance and high binding affinity, it seems likely that PARP-1 is the first factor to bind to DNA single-strand breaks in vivo (Fig. ), resulting in activation of its polymerase activity and automodification since PARP-1 itself is the major acceptor for PAR ( ).

    Techniques: Binding Assay, Activity Assay

    Identification of DNA ligase III-associated proteins by affinity chromatography. A HeLa nuclear extract was fractionated by using a DNA ligase III affinity chromatography column as described in Materials and Methods. (A) Proteins in comparable fractions eluted with 0.3 M NaCl from the beads liganded by GST and by GST-DNA ligase III (Lig III) were detected by silver staining after separation by SDS-PAGE. The polypeptides indicated were identified by MALDI-TOF mass spectrometry. The identities of PARP-1, Ku70, and Ku80 were verified by immunoblotting with PARP-1 (B) and Ku70 and Ku80 (C) monoclonal antibodies.

    Journal: Molecular and Cellular Biology

    Article Title: Physical and Functional Interaction between DNA Ligase III? and Poly(ADP-Ribose) Polymerase 1 in DNA Single-Strand Break Repair

    doi: 10.1128/MCB.23.16.5919-5927.2003

    Figure Lengend Snippet: Identification of DNA ligase III-associated proteins by affinity chromatography. A HeLa nuclear extract was fractionated by using a DNA ligase III affinity chromatography column as described in Materials and Methods. (A) Proteins in comparable fractions eluted with 0.3 M NaCl from the beads liganded by GST and by GST-DNA ligase III (Lig III) were detected by silver staining after separation by SDS-PAGE. The polypeptides indicated were identified by MALDI-TOF mass spectrometry. The identities of PARP-1, Ku70, and Ku80 were verified by immunoblotting with PARP-1 (B) and Ku70 and Ku80 (C) monoclonal antibodies.

    Article Snippet: Based on its relative abundance and high binding affinity, it seems likely that PARP-1 is the first factor to bind to DNA single-strand breaks in vivo (Fig. ), resulting in activation of its polymerase activity and automodification since PARP-1 itself is the major acceptor for PAR ( ).

    Techniques: Affinity Chromatography, Affinity Column, Silver Staining, SDS Page, Mass Spectrometry

    Binding of DNA ligase III and PARP-1 to DNA single-strand interruptions. Effects of NAD on DNA binding by PARP-1 are shown. (A) Analysis of the binding of DNA ligase III to a DNA single-strand break by surface plasmon resonance. (Top panel) Schematic representation of the nicked hairpin oligonucleotide immobilized on the chip surface. (Bottom panel) Representative sensorgram showing the binding and release of untagged full-length DNA ligase III (LigIII), injected at the concentrations indicated, from the nicked hairpin oligonucleotide immobilized on the chip surface. (B) Visualization of DNA ligase III bound to a DNA single-strand break by DNase I footprinting. Intact DNA ligase III (LigIII; 20 nM) was preincubated with the labeled, nicked DNA substrate (16 nM) indicated and then incubated with DNase I as described in Materials and Methods. After separation by denaturing gel electrophoresis, labeled oligonucleotides were detected by autoradiography. −, no enzyme. The vertical line indicates the region protected from DNase I activity. (C) Effect of DNA strand break binding by DNA ligase III and PARP-1 on T4 polynucleotide kinase activity. Intact DNA ligase III (150 nM) or PARP-1 (150 nM) was preincubated with the indicated DNA substrate containing a single-nucleotide (1 nt) gap in the presence (+) or absence (−) of NAD as indicated prior to incubation with polynucleotide kinase and [γ- 32 P]ATP as described in Materials and Methods. After separation by denaturing gel electrophoresis, labeled 29-mer oligonucleotide was detected and quantitated by phosphorimager analysis. Lane C, no PARP-1 or DNA ligase III.

    Journal: Molecular and Cellular Biology

    Article Title: Physical and Functional Interaction between DNA Ligase III? and Poly(ADP-Ribose) Polymerase 1 in DNA Single-Strand Break Repair

    doi: 10.1128/MCB.23.16.5919-5927.2003

    Figure Lengend Snippet: Binding of DNA ligase III and PARP-1 to DNA single-strand interruptions. Effects of NAD on DNA binding by PARP-1 are shown. (A) Analysis of the binding of DNA ligase III to a DNA single-strand break by surface plasmon resonance. (Top panel) Schematic representation of the nicked hairpin oligonucleotide immobilized on the chip surface. (Bottom panel) Representative sensorgram showing the binding and release of untagged full-length DNA ligase III (LigIII), injected at the concentrations indicated, from the nicked hairpin oligonucleotide immobilized on the chip surface. (B) Visualization of DNA ligase III bound to a DNA single-strand break by DNase I footprinting. Intact DNA ligase III (LigIII; 20 nM) was preincubated with the labeled, nicked DNA substrate (16 nM) indicated and then incubated with DNase I as described in Materials and Methods. After separation by denaturing gel electrophoresis, labeled oligonucleotides were detected by autoradiography. −, no enzyme. The vertical line indicates the region protected from DNase I activity. (C) Effect of DNA strand break binding by DNA ligase III and PARP-1 on T4 polynucleotide kinase activity. Intact DNA ligase III (150 nM) or PARP-1 (150 nM) was preincubated with the indicated DNA substrate containing a single-nucleotide (1 nt) gap in the presence (+) or absence (−) of NAD as indicated prior to incubation with polynucleotide kinase and [γ- 32 P]ATP as described in Materials and Methods. After separation by denaturing gel electrophoresis, labeled 29-mer oligonucleotide was detected and quantitated by phosphorimager analysis. Lane C, no PARP-1 or DNA ligase III.

    Article Snippet: Based on its relative abundance and high binding affinity, it seems likely that PARP-1 is the first factor to bind to DNA single-strand breaks in vivo (Fig. ), resulting in activation of its polymerase activity and automodification since PARP-1 itself is the major acceptor for PAR ( ).

    Techniques: Binding Assay, SPR Assay, Chromatin Immunoprecipitation, Injection, Footprinting, Labeling, Incubation, Nucleic Acid Electrophoresis, Autoradiography, Activity Assay

    Effect of DNA binding by PARP-1 on DNA joining. (A) Purified PARP-1 (200 nM) was preincubated with a labeled DNA duplex containing a single ligatable nick (50 nM) in the presence (+) or absence (−) of NAD. Subsequently, intact DNA ligase III (Lig III; 2.5 nM), a truncated version lacking the zinc finger (ΔZf-Lig III; 2.5 nM), or DNA ligase I (Lig I; 2.5 nM) was added to the reaction mixture as indicated. After incubation for 10 min at 25°C, labeled oligonucleotides were separated by denaturing gel electrophoresis. The labeled substrate (30-mer) and ligated product (50-mer) were detected and quantitated by phosphorimager analysis. (B) The results of three independent DNA joining assays are shown graphically. White bars, intact DNA ligase III; grey bars, truncated version of DNA ligase III lacking the zinc finger; black bars, DNA ligase I. (C) Purified PARP-1 (100 nM) was preincubated with a labeled DNA duplex containing a single ligatable nick (100 nM) in the presence (+) or absence (−) of NAD. After separation by SDS-PAGE, poly(ADP-ribosyl)ated PARP-1 was detected by immunoblotting (IB) with a monoclonal antibody against PAR. The positions of unmodified and poly(ADP-ribosyl)ated PARP-1 are indicated.

    Journal: Molecular and Cellular Biology

    Article Title: Physical and Functional Interaction between DNA Ligase III? and Poly(ADP-Ribose) Polymerase 1 in DNA Single-Strand Break Repair

    doi: 10.1128/MCB.23.16.5919-5927.2003

    Figure Lengend Snippet: Effect of DNA binding by PARP-1 on DNA joining. (A) Purified PARP-1 (200 nM) was preincubated with a labeled DNA duplex containing a single ligatable nick (50 nM) in the presence (+) or absence (−) of NAD. Subsequently, intact DNA ligase III (Lig III; 2.5 nM), a truncated version lacking the zinc finger (ΔZf-Lig III; 2.5 nM), or DNA ligase I (Lig I; 2.5 nM) was added to the reaction mixture as indicated. After incubation for 10 min at 25°C, labeled oligonucleotides were separated by denaturing gel electrophoresis. The labeled substrate (30-mer) and ligated product (50-mer) were detected and quantitated by phosphorimager analysis. (B) The results of three independent DNA joining assays are shown graphically. White bars, intact DNA ligase III; grey bars, truncated version of DNA ligase III lacking the zinc finger; black bars, DNA ligase I. (C) Purified PARP-1 (100 nM) was preincubated with a labeled DNA duplex containing a single ligatable nick (100 nM) in the presence (+) or absence (−) of NAD. After separation by SDS-PAGE, poly(ADP-ribosyl)ated PARP-1 was detected by immunoblotting (IB) with a monoclonal antibody against PAR. The positions of unmodified and poly(ADP-ribosyl)ated PARP-1 are indicated.

    Article Snippet: Based on its relative abundance and high binding affinity, it seems likely that PARP-1 is the first factor to bind to DNA single-strand breaks in vivo (Fig. ), resulting in activation of its polymerase activity and automodification since PARP-1 itself is the major acceptor for PAR ( ).

    Techniques: Binding Assay, Purification, Labeling, Incubation, Nucleic Acid Electrophoresis, SDS Page

    DNA ligase III preferentially binds to poly(ADP-ribosyl)ated PARP-1 in vitro. Effects of DNA damage on the association between PARP-1 and DNA ligase III-XRCC1 in vivo are shown. (A) Purified PARP-1 (500 nM) was preincubated with a labeled DNA duplex containing a single ligatable nick (300 nM) in the presence (+) or absence (−) of NAD. After treatment with DNase I, intact DNA ligase III (10 nM) or a truncated version lacking the zinc finger (10 nM) was added to the reaction mixture as indicated. Proteins immunoprecipitated (IP) by PARP-1 antibody were separated by SDS-PAGE and then detected by immunblotting (IB) with the indicated antibody. Upper panel, intact DNA ligase III (Lig III); middle panel, truncated version of DNA ligase III lacking the zinc finger (ΔZf-Lig III); lower panel, unmodified PARP-1 y(ADP-ribosyl)ated PARP-1 [P-(ADPR) n ]. (B) Effect of H 2 O 2 treatment on the association of PARP-1, DNA ligase IIIα, and XRCC1. Whole cell extracts (WCE) were prepared from undamaged (−) or damaged (+) HeLa cells as described in Materials and Methods. Equivalent aliquots of the cells to be damaged were pretreated with 1,5-isoquinolinediol (DiQ; 100 μM) as indicated for 1 h prior to and during H 2 O 2 treatment. Proteins immunoprecipitated by PARP-1 antibody were separated by SDS-PAGE and then detected by immunoblotting with the indicated antibody. DNA ligase IIIα and XRCC1 in the extracts from undamaged cells were detected by direct immunoblotting.

    Journal: Molecular and Cellular Biology

    Article Title: Physical and Functional Interaction between DNA Ligase III? and Poly(ADP-Ribose) Polymerase 1 in DNA Single-Strand Break Repair

    doi: 10.1128/MCB.23.16.5919-5927.2003

    Figure Lengend Snippet: DNA ligase III preferentially binds to poly(ADP-ribosyl)ated PARP-1 in vitro. Effects of DNA damage on the association between PARP-1 and DNA ligase III-XRCC1 in vivo are shown. (A) Purified PARP-1 (500 nM) was preincubated with a labeled DNA duplex containing a single ligatable nick (300 nM) in the presence (+) or absence (−) of NAD. After treatment with DNase I, intact DNA ligase III (10 nM) or a truncated version lacking the zinc finger (10 nM) was added to the reaction mixture as indicated. Proteins immunoprecipitated (IP) by PARP-1 antibody were separated by SDS-PAGE and then detected by immunblotting (IB) with the indicated antibody. Upper panel, intact DNA ligase III (Lig III); middle panel, truncated version of DNA ligase III lacking the zinc finger (ΔZf-Lig III); lower panel, unmodified PARP-1 y(ADP-ribosyl)ated PARP-1 [P-(ADPR) n ]. (B) Effect of H 2 O 2 treatment on the association of PARP-1, DNA ligase IIIα, and XRCC1. Whole cell extracts (WCE) were prepared from undamaged (−) or damaged (+) HeLa cells as described in Materials and Methods. Equivalent aliquots of the cells to be damaged were pretreated with 1,5-isoquinolinediol (DiQ; 100 μM) as indicated for 1 h prior to and during H 2 O 2 treatment. Proteins immunoprecipitated by PARP-1 antibody were separated by SDS-PAGE and then detected by immunoblotting with the indicated antibody. DNA ligase IIIα and XRCC1 in the extracts from undamaged cells were detected by direct immunoblotting.

    Article Snippet: Based on its relative abundance and high binding affinity, it seems likely that PARP-1 is the first factor to bind to DNA single-strand breaks in vivo (Fig. ), resulting in activation of its polymerase activity and automodification since PARP-1 itself is the major acceptor for PAR ( ).

    Techniques: In Vitro, In Vivo, Purification, Labeling, Immunoprecipitation, SDS Page

    Inhibition of PARP1 promoted the transcription of FXR-dependent hepatoprotective genes. (A and B) Real-time RT-PCR assays of BSEP , FGF19 , Foxm1b , and SHP in HepG2 cells. After treatment with 1 μM GW4064 (A) or 15 μM PJ34 (B) for 12 h, cells were treated or not treated with H 2 O 2 (300 μM; 12 h). Data are expressed as means ± SEM. Significant differences from the control group (**, P

    Journal: Molecular and Cellular Biology

    Article Title: Poly(ADP-Ribose) Polymerase 1 Promotes Oxidative-Stress-Induced Liver Cell Death via Suppressing Farnesoid X Receptor ?

    doi: 10.1128/MCB.00160-13

    Figure Lengend Snippet: Inhibition of PARP1 promoted the transcription of FXR-dependent hepatoprotective genes. (A and B) Real-time RT-PCR assays of BSEP , FGF19 , Foxm1b , and SHP in HepG2 cells. After treatment with 1 μM GW4064 (A) or 15 μM PJ34 (B) for 12 h, cells were treated or not treated with H 2 O 2 (300 μM; 12 h). Data are expressed as means ± SEM. Significant differences from the control group (**, P

    Article Snippet: Given that PARP1 is known as a poly(ADP-ribosyl)transferase, we then speculated that the regulatory effects of PARP1 on the transactivation of FXR might be based on the enzymatic activity of PARP1.

    Techniques: Inhibition, Quantitative RT-PCR

    Poly(ADP-ribosyl)ation inhibited the binding of FXR to FXRE in the target promoter. (A) HepG2 cells were treated with 15 μM PJ34 for 24 h with or without H 2 O 2 treatment (300 μM; 12 h). Binding of FXR to FXRE was detected by EMSA. (B) HepG2 cells were transfected with either PARP1 siRNA or an unrelated siRNA at 50 nM for 48 h, with or without H 2 O 2 treatment (300 μM; 12 h). Binding of FXR to FXRE was detected by EMSA. (C) Nuclear extracts from untreated HepG2 cells were incubated with active DNA and NAD + (1, 10, or 100 μM) and were then subjected to EMSA. (D and E) ChIP-PCR assays using an anti-FXR antibody for amplification of BSEP promoters in HepG2 cells treated with 15 μM PJ34 for 24 h (D) or transfected with PARP1 siRNA or an unrelated siRNA at 50 nM for 48 h (E), with or without H 2 O 2 treatment (300 μM; 12 h). Data are expressed as means ± SEM. Significant differences from the control group (**, P

    Journal: Molecular and Cellular Biology

    Article Title: Poly(ADP-Ribose) Polymerase 1 Promotes Oxidative-Stress-Induced Liver Cell Death via Suppressing Farnesoid X Receptor ?

    doi: 10.1128/MCB.00160-13

    Figure Lengend Snippet: Poly(ADP-ribosyl)ation inhibited the binding of FXR to FXRE in the target promoter. (A) HepG2 cells were treated with 15 μM PJ34 for 24 h with or without H 2 O 2 treatment (300 μM; 12 h). Binding of FXR to FXRE was detected by EMSA. (B) HepG2 cells were transfected with either PARP1 siRNA or an unrelated siRNA at 50 nM for 48 h, with or without H 2 O 2 treatment (300 μM; 12 h). Binding of FXR to FXRE was detected by EMSA. (C) Nuclear extracts from untreated HepG2 cells were incubated with active DNA and NAD + (1, 10, or 100 μM) and were then subjected to EMSA. (D and E) ChIP-PCR assays using an anti-FXR antibody for amplification of BSEP promoters in HepG2 cells treated with 15 μM PJ34 for 24 h (D) or transfected with PARP1 siRNA or an unrelated siRNA at 50 nM for 48 h (E), with or without H 2 O 2 treatment (300 μM; 12 h). Data are expressed as means ± SEM. Significant differences from the control group (**, P

    Article Snippet: Given that PARP1 is known as a poly(ADP-ribosyl)transferase, we then speculated that the regulatory effects of PARP1 on the transactivation of FXR might be based on the enzymatic activity of PARP1.

    Techniques: Binding Assay, Transfection, Incubation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification

    PARP1 bound directly to FXR. (A) Far-Western blot assays of nuclear extracts from HepG2 cells treated with an unrelated siRNA or FXR siRNA. Unpoly(ADP-ribosyl)ated PARP1 (UP-PARP1), autopoly(ADP-ribosyl)ated PARP1 (AP-PARP1), or β-actin protein (negative control) was used as a probe. Histone H1 (his1) served as a loading control (bottom panels). (B) Coimmunoprecipitation assays of FXR-bound proteins from HepG2 cells, followed by Western blot assays using an anti-PARP1 antibody. Nonspecific IgG served as a negative control. (C) Coimmunoprecipitation assays of PARP1-bound proteins or poly(ADP-ribosyl)ated proteins from HepG2 cells, followed by Western blot assays using an anti-FXR antibody. Nonspecific IgG served as a negative control. (D) Far-Western blot assays of recombinant FXR protein. UP-PARP1 or AP-PARP1 was used as a probe. β-Actin protein served as a negative control. (E) Diagram of Flag-tagged human PARP1 with its domains: DNA-binding domain (DBD), nuclear localization signal (NLS), BRCA1 C terminus (BRCT)/automodification domain (AMD), and catalytic domain (CD). Fragments A to F with their amino acid coordinates are listed. HepG2 cells were transfected with EGFP-tagged full-length FXR and Flag-tagged PARP1 mutants. Coimmunoprecipitation assays demonstrated the specific binding of FXR to the BRCT/AMD of PARP1. (F) Diagram of EGFP-tagged human FXR with its domains. LBD, ligand-binding domain. Fragments A to E with their amino acid coordinates are listed. HepG2 cells were transfected with Flag-tagged full-length PARP1 and EGFP-tagged FXR mutants. Coimmunoprecipitation assays demonstrated the specific binding of PARP1 to the LBD of FXR.

    Journal: Molecular and Cellular Biology

    Article Title: Poly(ADP-Ribose) Polymerase 1 Promotes Oxidative-Stress-Induced Liver Cell Death via Suppressing Farnesoid X Receptor ?

    doi: 10.1128/MCB.00160-13

    Figure Lengend Snippet: PARP1 bound directly to FXR. (A) Far-Western blot assays of nuclear extracts from HepG2 cells treated with an unrelated siRNA or FXR siRNA. Unpoly(ADP-ribosyl)ated PARP1 (UP-PARP1), autopoly(ADP-ribosyl)ated PARP1 (AP-PARP1), or β-actin protein (negative control) was used as a probe. Histone H1 (his1) served as a loading control (bottom panels). (B) Coimmunoprecipitation assays of FXR-bound proteins from HepG2 cells, followed by Western blot assays using an anti-PARP1 antibody. Nonspecific IgG served as a negative control. (C) Coimmunoprecipitation assays of PARP1-bound proteins or poly(ADP-ribosyl)ated proteins from HepG2 cells, followed by Western blot assays using an anti-FXR antibody. Nonspecific IgG served as a negative control. (D) Far-Western blot assays of recombinant FXR protein. UP-PARP1 or AP-PARP1 was used as a probe. β-Actin protein served as a negative control. (E) Diagram of Flag-tagged human PARP1 with its domains: DNA-binding domain (DBD), nuclear localization signal (NLS), BRCA1 C terminus (BRCT)/automodification domain (AMD), and catalytic domain (CD). Fragments A to F with their amino acid coordinates are listed. HepG2 cells were transfected with EGFP-tagged full-length FXR and Flag-tagged PARP1 mutants. Coimmunoprecipitation assays demonstrated the specific binding of FXR to the BRCT/AMD of PARP1. (F) Diagram of EGFP-tagged human FXR with its domains. LBD, ligand-binding domain. Fragments A to E with their amino acid coordinates are listed. HepG2 cells were transfected with Flag-tagged full-length PARP1 and EGFP-tagged FXR mutants. Coimmunoprecipitation assays demonstrated the specific binding of PARP1 to the LBD of FXR.

    Article Snippet: Given that PARP1 is known as a poly(ADP-ribosyl)transferase, we then speculated that the regulatory effects of PARP1 on the transactivation of FXR might be based on the enzymatic activity of PARP1.

    Techniques: Far Western Blot, Negative Control, Western Blot, Recombinant, Binding Assay, Transfection, Ligand Binding Assay

    Oxidative stress-induced HepG2 cell death was attenuated by PARP1 inhibition. (A) HepG2 cells were exposed to H 2 O 2 (3, 30, or 300 μM) for 12 h. Cells were trypsinized and were then counted in 3 fields by a hemocytometer with trypan blue dye ( n = 3). Asterisks indicate significant differences (*, P

    Journal: Molecular and Cellular Biology

    Article Title: Poly(ADP-Ribose) Polymerase 1 Promotes Oxidative-Stress-Induced Liver Cell Death via Suppressing Farnesoid X Receptor ?

    doi: 10.1128/MCB.00160-13

    Figure Lengend Snippet: Oxidative stress-induced HepG2 cell death was attenuated by PARP1 inhibition. (A) HepG2 cells were exposed to H 2 O 2 (3, 30, or 300 μM) for 12 h. Cells were trypsinized and were then counted in 3 fields by a hemocytometer with trypan blue dye ( n = 3). Asterisks indicate significant differences (*, P

    Article Snippet: Given that PARP1 is known as a poly(ADP-ribosyl)transferase, we then speculated that the regulatory effects of PARP1 on the transactivation of FXR might be based on the enzymatic activity of PARP1.

    Techniques: Inhibition

    PARP1 poly(ADP-ribosyl)ated the LBD of FXR. (A) Nuclear extracts from HepG2 cells were subjected to an immunoprecipitation assay with an anti-FXR antibody, followed by a Western blot assay using an anti-PAR antibody. Cells were treated with 15 μM PJ34 for 24 h in the presence or absence of H 2 O 2 (300 μM; 12 h). (B) Nuclear extracts from HepG2 cells were subjected to an immunoprecipitation assay with an anti-FXR antibody, followed by a Western blot assay using an anti-PAR antibody. Cells were transfected with PARP1 siRNA or unrelated siRNA at 50 nM for 48 h with or without H 2 O 2 treatment (300 μM; 12 h). (C) Recombinant FXR proteins were incubated either with a vehicle (PBS), with PARP1, NAD + , and active DNA, or with PARP1, NAD + , active DNA, and 3AB, as indicated. Western blot assays were used to detect the poly(ADP-ribosyl)ation levels of FXR. (D) (Top) Diagram of GST-tagged human FXR with its domains. (Center) Purified GST-FXR fragments are shown after Coomassie staining. (Bottom) Bacterially expressed GST-FXR deletion mutants were incubated with recombinant PARP1 protein in the presence of DNA and NAD + . Poly(ADP-ribosyl)ation of GST-FXR mutants was detected by a Western blot assay with an anti-PAR antibody.

    Journal: Molecular and Cellular Biology

    Article Title: Poly(ADP-Ribose) Polymerase 1 Promotes Oxidative-Stress-Induced Liver Cell Death via Suppressing Farnesoid X Receptor ?

    doi: 10.1128/MCB.00160-13

    Figure Lengend Snippet: PARP1 poly(ADP-ribosyl)ated the LBD of FXR. (A) Nuclear extracts from HepG2 cells were subjected to an immunoprecipitation assay with an anti-FXR antibody, followed by a Western blot assay using an anti-PAR antibody. Cells were treated with 15 μM PJ34 for 24 h in the presence or absence of H 2 O 2 (300 μM; 12 h). (B) Nuclear extracts from HepG2 cells were subjected to an immunoprecipitation assay with an anti-FXR antibody, followed by a Western blot assay using an anti-PAR antibody. Cells were transfected with PARP1 siRNA or unrelated siRNA at 50 nM for 48 h with or without H 2 O 2 treatment (300 μM; 12 h). (C) Recombinant FXR proteins were incubated either with a vehicle (PBS), with PARP1, NAD + , and active DNA, or with PARP1, NAD + , active DNA, and 3AB, as indicated. Western blot assays were used to detect the poly(ADP-ribosyl)ation levels of FXR. (D) (Top) Diagram of GST-tagged human FXR with its domains. (Center) Purified GST-FXR fragments are shown after Coomassie staining. (Bottom) Bacterially expressed GST-FXR deletion mutants were incubated with recombinant PARP1 protein in the presence of DNA and NAD + . Poly(ADP-ribosyl)ation of GST-FXR mutants was detected by a Western blot assay with an anti-PAR antibody.

    Article Snippet: Given that PARP1 is known as a poly(ADP-ribosyl)transferase, we then speculated that the regulatory effects of PARP1 on the transactivation of FXR might be based on the enzymatic activity of PARP1.

    Techniques: Immunoprecipitation, Western Blot, Transfection, Recombinant, Incubation, Purification, Staining

    Ligand-induced FXR transactivation was mediated by the inhibition of poly(ADP-ribosyl)ation. (A) Nuclear extracts from HepG2 cells were subjected to an immunoprecipitation assay with an anti-FXR antibody, followed by a Western blot assay using an anti-PAR antibody. Cells were treated with GW4064 (0.5, 1, or 2 μM) or a vehicle (dimethyl sulfoxide) for 24 h. (B) Nuclear extracts from HepG2 cells were subjected to an immunoprecipitation assay with an anti-FXR antibody, followed by a Western blot assay using an anti-PAR antibody. Cells were treated with 1 μM GW4064 for 24 h, with or without H 2 O 2 (300 μM; 12 h). (C) The protein expression of PARP1 or FXR was determined by a Western blot assay. HepG2 cells were treated with GW4064 (0.5, 1, or 2 μM) or a vehicle (dimethyl sulfoxide) for 24 h. (D) EMSAs were used to detect the FXR-FXRE complex in HepG2 cells. After treatment with an empty vector (p3flag-CMV), wt-PARP1, or mut-PARP1 at 1 mg/liter for 24 h, cells were treated with nicotinic acid (50 μM; 24 h), GW4064 (1 μM; 24 h), or CDCA (50 μM; 24 h) as indicated. Nicotinic acid served as a negative control. (E) ChIP-PCR assays using an anti-FXR antibody for the amplification of BSEP promoters. HepG2 cells were treated with an empty vector (p3flag-CMV), wt-PARP1, or mut-PARP1 at 1 mg/liter for 48 h in the absence or presence of nicotinic acid (50 μM; 24 h), GW4064 (1 μM; 24 h), or CDCA (50 μM; 24 h) as indicated. Data are expressed as means ± SEM. Significant differences from the control group (*, P

    Journal: Molecular and Cellular Biology

    Article Title: Poly(ADP-Ribose) Polymerase 1 Promotes Oxidative-Stress-Induced Liver Cell Death via Suppressing Farnesoid X Receptor ?

    doi: 10.1128/MCB.00160-13

    Figure Lengend Snippet: Ligand-induced FXR transactivation was mediated by the inhibition of poly(ADP-ribosyl)ation. (A) Nuclear extracts from HepG2 cells were subjected to an immunoprecipitation assay with an anti-FXR antibody, followed by a Western blot assay using an anti-PAR antibody. Cells were treated with GW4064 (0.5, 1, or 2 μM) or a vehicle (dimethyl sulfoxide) for 24 h. (B) Nuclear extracts from HepG2 cells were subjected to an immunoprecipitation assay with an anti-FXR antibody, followed by a Western blot assay using an anti-PAR antibody. Cells were treated with 1 μM GW4064 for 24 h, with or without H 2 O 2 (300 μM; 12 h). (C) The protein expression of PARP1 or FXR was determined by a Western blot assay. HepG2 cells were treated with GW4064 (0.5, 1, or 2 μM) or a vehicle (dimethyl sulfoxide) for 24 h. (D) EMSAs were used to detect the FXR-FXRE complex in HepG2 cells. After treatment with an empty vector (p3flag-CMV), wt-PARP1, or mut-PARP1 at 1 mg/liter for 24 h, cells were treated with nicotinic acid (50 μM; 24 h), GW4064 (1 μM; 24 h), or CDCA (50 μM; 24 h) as indicated. Nicotinic acid served as a negative control. (E) ChIP-PCR assays using an anti-FXR antibody for the amplification of BSEP promoters. HepG2 cells were treated with an empty vector (p3flag-CMV), wt-PARP1, or mut-PARP1 at 1 mg/liter for 48 h in the absence or presence of nicotinic acid (50 μM; 24 h), GW4064 (1 μM; 24 h), or CDCA (50 μM; 24 h) as indicated. Data are expressed as means ± SEM. Significant differences from the control group (*, P

    Article Snippet: Given that PARP1 is known as a poly(ADP-ribosyl)transferase, we then speculated that the regulatory effects of PARP1 on the transactivation of FXR might be based on the enzymatic activity of PARP1.

    Techniques: Inhibition, Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation, Negative Control, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification

    Fatostatin inhibits growth of and induces EnRS in MCF-7 xenograft tumors. MCF-7 cell xenograft tumors were initiated in athymic mice supplemented with estradiol capsules. Once tumors reached ~25 mm 2 , FS or DMSO control were administered daily ( n = 12–14 tumors/group). a Tumor size was measured and plotted over time. b Tumors were excised after 16 days of treatment and weighed. Animal body weight on day 16 is indicated. c–e Ki67 and cleaved PARP were examined in FFPE tumor sections by IHC and quantified. Bars represent 100 µm. f p-eIF2α was examined by IF with DAPI as a nuclear stain in DMSO and FS-treated tumors. g Quantitation of immunofluorescence was performed using the cell seed/spot segmentation analysis in ImageJ FIJI. The number of cells with color intensity +5% over background were counted as positive staining for p-eIF2α. h The mean color intensity of each cell staining positive for p-eIF2α was determined and plotted as number of cells vs. intensity. i SREBP1 was examined by IF with DAPI as a nuclear stain in DMSO and FS-treated tumors. j The number of nuclei with SREBP1 staining was determined and plotted per 100 cells. * P

    Journal: Oncogenesis

    Article Title: Fatostatin induces pro- and anti-apoptotic lipid accumulation in breast cancer

    doi: 10.1038/s41389-018-0076-0

    Figure Lengend Snippet: Fatostatin inhibits growth of and induces EnRS in MCF-7 xenograft tumors. MCF-7 cell xenograft tumors were initiated in athymic mice supplemented with estradiol capsules. Once tumors reached ~25 mm 2 , FS or DMSO control were administered daily ( n = 12–14 tumors/group). a Tumor size was measured and plotted over time. b Tumors were excised after 16 days of treatment and weighed. Animal body weight on day 16 is indicated. c–e Ki67 and cleaved PARP were examined in FFPE tumor sections by IHC and quantified. Bars represent 100 µm. f p-eIF2α was examined by IF with DAPI as a nuclear stain in DMSO and FS-treated tumors. g Quantitation of immunofluorescence was performed using the cell seed/spot segmentation analysis in ImageJ FIJI. The number of cells with color intensity +5% over background were counted as positive staining for p-eIF2α. h The mean color intensity of each cell staining positive for p-eIF2α was determined and plotted as number of cells vs. intensity. i SREBP1 was examined by IF with DAPI as a nuclear stain in DMSO and FS-treated tumors. j The number of nuclei with SREBP1 staining was determined and plotted per 100 cells. * P

    Article Snippet: Antibodies against SREBP1, PARP, and cleaved-PARP were purchased from Abcam (#ab28481; #ab32138; #ab32064; #ab150064; #ab150077).

    Techniques: Mouse Assay, Formalin-fixed Paraffin-Embedded, Immunohistochemistry, Staining, Quantitation Assay, Immunofluorescence

    Targeting of RAD51 activates several signaling pathways but attenuated by combining with PARP and p38 inhibition A. The Human Phospho-Kinase arrays (R D Systems) were probed with MDA-MB-231 lysate samples that had been treated for 72 hours; samples used are labeled (from top to bottom): (1) DMSO treatment, (2) 10 μM RAD51i, (3) 2.5 μM PARPi/10 μM p38i, and (4) 10 μM RAD51i /2.5 μM PARPi/10 μM p38i triple combination. Highlighted dots represent a significant change in signal over DMSO treated controls. The corners are positive control blots for quantification. B. Quantitation of spot intensity was standardized for cells treated with DMSO and plotted as normalized intensity. Several kinases displayed greater than 2 fold increase in phosphorylation compared to references. Shading represents grouping based on pathway signaling. C. Protein expression and changes in phosphorylation of ERK1/2, p38, STAT3, MK-2 (p38 target) and AKT were confirmed by western blotting.

    Journal: Oncotarget

    Article Title: RAD51 inhibition in triple negative breast cancer cells is challenged by compensatory survival signaling and requires rational combination therapy

    doi: 10.18632/oncotarget.11065

    Figure Lengend Snippet: Targeting of RAD51 activates several signaling pathways but attenuated by combining with PARP and p38 inhibition A. The Human Phospho-Kinase arrays (R D Systems) were probed with MDA-MB-231 lysate samples that had been treated for 72 hours; samples used are labeled (from top to bottom): (1) DMSO treatment, (2) 10 μM RAD51i, (3) 2.5 μM PARPi/10 μM p38i, and (4) 10 μM RAD51i /2.5 μM PARPi/10 μM p38i triple combination. Highlighted dots represent a significant change in signal over DMSO treated controls. The corners are positive control blots for quantification. B. Quantitation of spot intensity was standardized for cells treated with DMSO and plotted as normalized intensity. Several kinases displayed greater than 2 fold increase in phosphorylation compared to references. Shading represents grouping based on pathway signaling. C. Protein expression and changes in phosphorylation of ERK1/2, p38, STAT3, MK-2 (p38 target) and AKT were confirmed by western blotting.

    Article Snippet: Immunoblots were probed with anti-RAD51 (Santa Cruz Biotech), anti-p38, anti-phospho-p38 (Cell Signaling), anti-PARP (BD Biosciences), anti-MK2 (Cell Signaling), anti-HSP27, anti-phospho-HSP27 (Cell Signaling), anti-ERK1/2, anti-phospho-ERK1/2 (Cell Signaling) and anti-Δ-Actin, (Sigma) as a loading control.

    Techniques: Inhibition, Multiple Displacement Amplification, Labeling, Positive Control, Quantitation Assay, Expressing, Western Blot

    The combination of RAD51/PARP/p38 inhibition retards TNBC growth in vitro A. MDA-MB-231 cells were incubated in the presence of single, dual or triple drug combinations using 10 μM RAD51i, 10 μM p38i and 2.5 μM PARPi and cell growth followed over 72 hours. Results are average percentage confluency of the well (* p

    Journal: Oncotarget

    Article Title: RAD51 inhibition in triple negative breast cancer cells is challenged by compensatory survival signaling and requires rational combination therapy

    doi: 10.18632/oncotarget.11065

    Figure Lengend Snippet: The combination of RAD51/PARP/p38 inhibition retards TNBC growth in vitro A. MDA-MB-231 cells were incubated in the presence of single, dual or triple drug combinations using 10 μM RAD51i, 10 μM p38i and 2.5 μM PARPi and cell growth followed over 72 hours. Results are average percentage confluency of the well (* p

    Article Snippet: Immunoblots were probed with anti-RAD51 (Santa Cruz Biotech), anti-p38, anti-phospho-p38 (Cell Signaling), anti-PARP (BD Biosciences), anti-MK2 (Cell Signaling), anti-HSP27, anti-phospho-HSP27 (Cell Signaling), anti-ERK1/2, anti-phospho-ERK1/2 (Cell Signaling) and anti-Δ-Actin, (Sigma) as a loading control.

    Techniques: Inhibition, In Vitro, Multiple Displacement Amplification, Incubation

    RAD51, PARP and p38 combined inhibition in vivo A. MDA-MB-231 xenografts were monitored by bioluminescence (luciferin) imaging, DMSO and triple therapy cohorts are represented. B. Tumor burden plotted as percentage change in volume for all single arm controls, double combinations and triple therapy combination compared to days after treatment with all three drugs. *p

    Journal: Oncotarget

    Article Title: RAD51 inhibition in triple negative breast cancer cells is challenged by compensatory survival signaling and requires rational combination therapy

    doi: 10.18632/oncotarget.11065

    Figure Lengend Snippet: RAD51, PARP and p38 combined inhibition in vivo A. MDA-MB-231 xenografts were monitored by bioluminescence (luciferin) imaging, DMSO and triple therapy cohorts are represented. B. Tumor burden plotted as percentage change in volume for all single arm controls, double combinations and triple therapy combination compared to days after treatment with all three drugs. *p

    Article Snippet: Immunoblots were probed with anti-RAD51 (Santa Cruz Biotech), anti-p38, anti-phospho-p38 (Cell Signaling), anti-PARP (BD Biosciences), anti-MK2 (Cell Signaling), anti-HSP27, anti-phospho-HSP27 (Cell Signaling), anti-ERK1/2, anti-phospho-ERK1/2 (Cell Signaling) and anti-Δ-Actin, (Sigma) as a loading control.

    Techniques: Inhibition, In Vivo, Multiple Displacement Amplification, Imaging

    Combination of RAD51, PARP and p38 inhibitors against TNBC cell lines The three TNBC cell lines MDA-MB-231 (top row), MDA-MB-436 (middle row) and PMC42-ET (bottom row) were used for dose response studies using three molecular inhibitors targeting RAD51 (B02, RAD51i), PARP (ABT-888, PARPi) and p38 (LY2228820, p38i). Dose curves for the single drugs were performed using 0-100 μM escalating doses. The combinations were carried out as follows: A–C. RAD51i escalating doses 0-100 μM with 2.5 μM of PARPi; D–F. RAD51i escalating doses 0-100 μM with 10 μM of p38i; G–I. p38i escalating doses 0-100 μM with 2.5 μM of PARPi J–L. RAD51i escalating doses 0-100 μM with 2.5 μM PARPi alone or with 10 μM of p38i. All experiments were performed over 72 hours in triplicate and graphs represent line of best-fit non-linear regression +/−SEM.

    Journal: Oncotarget

    Article Title: RAD51 inhibition in triple negative breast cancer cells is challenged by compensatory survival signaling and requires rational combination therapy

    doi: 10.18632/oncotarget.11065

    Figure Lengend Snippet: Combination of RAD51, PARP and p38 inhibitors against TNBC cell lines The three TNBC cell lines MDA-MB-231 (top row), MDA-MB-436 (middle row) and PMC42-ET (bottom row) were used for dose response studies using three molecular inhibitors targeting RAD51 (B02, RAD51i), PARP (ABT-888, PARPi) and p38 (LY2228820, p38i). Dose curves for the single drugs were performed using 0-100 μM escalating doses. The combinations were carried out as follows: A–C. RAD51i escalating doses 0-100 μM with 2.5 μM of PARPi; D–F. RAD51i escalating doses 0-100 μM with 10 μM of p38i; G–I. p38i escalating doses 0-100 μM with 2.5 μM of PARPi J–L. RAD51i escalating doses 0-100 μM with 2.5 μM PARPi alone or with 10 μM of p38i. All experiments were performed over 72 hours in triplicate and graphs represent line of best-fit non-linear regression +/−SEM.

    Article Snippet: Immunoblots were probed with anti-RAD51 (Santa Cruz Biotech), anti-p38, anti-phospho-p38 (Cell Signaling), anti-PARP (BD Biosciences), anti-MK2 (Cell Signaling), anti-HSP27, anti-phospho-HSP27 (Cell Signaling), anti-ERK1/2, anti-phospho-ERK1/2 (Cell Signaling) and anti-Δ-Actin, (Sigma) as a loading control.

    Techniques: Multiple Displacement Amplification

    Schematic illustration of the impact of PARP-1 loss of function leading to EMT induction and aggressive prostate tumor growth, potentially mediated by Smad-directed TGF-β signaling. A potential cross-effect by nuclear AR depletion may contribute to enhanced EMT.

    Journal: Carcinogenesis

    Article Title: PARP-1 regulates epithelial–mesenchymal transition (EMT) in prostate tumorigenesis

    doi: 10.1093/carcin/bgu183

    Figure Lengend Snippet: Schematic illustration of the impact of PARP-1 loss of function leading to EMT induction and aggressive prostate tumor growth, potentially mediated by Smad-directed TGF-β signaling. A potential cross-effect by nuclear AR depletion may contribute to enhanced EMT.

    Article Snippet: Animals (3–6 mice/group) were divided into the following experimental groups: (i) TRAMP−/−, PARP-1−/−; (ii) TRAMP−/−, PARP-1+/−; (iii) TRAMP−/−, PARP-1+/+; (iv) TRAMP+/−, PARP-1−/−; (v) TRAMP+/−, PARP-1+/−; (vi) TRAMP+/−, PARP-1+/+.

    Techniques:

    Dysfunctional PARP-1 induces TGF-β-mediated EMT in prostate tumors. Analysis of expression of TGF-β signaling effectors by immunostaining and western blotting in prostate tissue from TRAMP mice harboring PARP-1 functional loss compared with TRAMP+/− PARP-1+/+ mice ( A and B , respectively). ( A ) Reveals intense nuclear immunoreactivity for the TGF-β intracellular effector, Smad3 protein in prostate tumor cells in tissue specimens from TRAMP+/− PARP-1−/− mice, compared with age-matched TRAMP+/− PARP-1+/+ mice; magnification, ×400. ( B ) Comparative protein profiling by western blot analysis of prostate tumor lysates for TGF-β (ligand) and its main intracellular effectors Smad 3 and Smad4 in TRAMP+/− PARP-1−/− prostate tumors versus WT mice, and Snail, EMT regulator, were also detected by western blotting. Results are shown for each of the three independent groups of mice analyzed (for each genotype) are shown (groups I, II and III). Molecular weights are shown on the right (kDa) for each specific protein. ( C ) Indicates the results of densitometric analysis from B, with the barographs showing the average value of band intensity relative to the loading control protein.

    Journal: Carcinogenesis

    Article Title: PARP-1 regulates epithelial–mesenchymal transition (EMT) in prostate tumorigenesis

    doi: 10.1093/carcin/bgu183

    Figure Lengend Snippet: Dysfunctional PARP-1 induces TGF-β-mediated EMT in prostate tumors. Analysis of expression of TGF-β signaling effectors by immunostaining and western blotting in prostate tissue from TRAMP mice harboring PARP-1 functional loss compared with TRAMP+/− PARP-1+/+ mice ( A and B , respectively). ( A ) Reveals intense nuclear immunoreactivity for the TGF-β intracellular effector, Smad3 protein in prostate tumor cells in tissue specimens from TRAMP+/− PARP-1−/− mice, compared with age-matched TRAMP+/− PARP-1+/+ mice; magnification, ×400. ( B ) Comparative protein profiling by western blot analysis of prostate tumor lysates for TGF-β (ligand) and its main intracellular effectors Smad 3 and Smad4 in TRAMP+/− PARP-1−/− prostate tumors versus WT mice, and Snail, EMT regulator, were also detected by western blotting. Results are shown for each of the three independent groups of mice analyzed (for each genotype) are shown (groups I, II and III). Molecular weights are shown on the right (kDa) for each specific protein. ( C ) Indicates the results of densitometric analysis from B, with the barographs showing the average value of band intensity relative to the loading control protein.

    Article Snippet: Animals (3–6 mice/group) were divided into the following experimental groups: (i) TRAMP−/−, PARP-1−/−; (ii) TRAMP−/−, PARP-1+/−; (iii) TRAMP−/−, PARP-1+/+; (iv) TRAMP+/−, PARP-1−/−; (v) TRAMP+/−, PARP-1+/−; (vi) TRAMP+/−, PARP-1+/+.

    Techniques: Expressing, Immunostaining, Western Blot, Mouse Assay, Functional Assay

    PARP-1 functional loss in TRAMP model increases prostate tumor aggressiveness via enhanced proliferation and reduced apoptosis. ( A ) Histological evaluation of prostate tissue from TRAMP+/− PARP-1+/+ and TRAMP+/− PARP-1−/− mice of increasing age (16, 20, 24 and 28 weeks) by hematoxylin and eosin staining. ( B ) Prostate cell proliferative index in tumors from TRAMP+/− PARP-1+/+ and TRAMP+/− PARP-1−/− mice. Ki-67 immunoreactivity was significantly higher in prostate tissue from TRAMP+/− PARP-1−/− compared with TRAMP+/− PARP-1+/+ mice; magnification ×400. (D) Quantitative analysis of Ki-67 staining. (*) indicates statistical significance at P

    Journal: Carcinogenesis

    Article Title: PARP-1 regulates epithelial–mesenchymal transition (EMT) in prostate tumorigenesis

    doi: 10.1093/carcin/bgu183

    Figure Lengend Snippet: PARP-1 functional loss in TRAMP model increases prostate tumor aggressiveness via enhanced proliferation and reduced apoptosis. ( A ) Histological evaluation of prostate tissue from TRAMP+/− PARP-1+/+ and TRAMP+/− PARP-1−/− mice of increasing age (16, 20, 24 and 28 weeks) by hematoxylin and eosin staining. ( B ) Prostate cell proliferative index in tumors from TRAMP+/− PARP-1+/+ and TRAMP+/− PARP-1−/− mice. Ki-67 immunoreactivity was significantly higher in prostate tissue from TRAMP+/− PARP-1−/− compared with TRAMP+/− PARP-1+/+ mice; magnification ×400. (D) Quantitative analysis of Ki-67 staining. (*) indicates statistical significance at P

    Article Snippet: Animals (3–6 mice/group) were divided into the following experimental groups: (i) TRAMP−/−, PARP-1−/−; (ii) TRAMP−/−, PARP-1+/−; (iii) TRAMP−/−, PARP-1+/+; (iv) TRAMP+/−, PARP-1−/−; (v) TRAMP+/−, PARP-1+/−; (vi) TRAMP+/−, PARP-1+/+.

    Techniques: Functional Assay, Mouse Assay, Staining

    Loss of PARP-1 reduces AR nuclear localization and activity. ( A ) Profiling of AR protein localization and immunoreactivity in prostate tissues from TRAMP+/− PARP+/+ and TRAMP+/− PARP-1−/− mice (20 weeks); magnification ×400. ( B ) Quantitative analysis of data from A, reveals a significant decrease in nuclear AR in prostate tissue from TRAMP+/−, PARP-1−/− mice compared with controls ( P

    Journal: Carcinogenesis

    Article Title: PARP-1 regulates epithelial–mesenchymal transition (EMT) in prostate tumorigenesis

    doi: 10.1093/carcin/bgu183

    Figure Lengend Snippet: Loss of PARP-1 reduces AR nuclear localization and activity. ( A ) Profiling of AR protein localization and immunoreactivity in prostate tissues from TRAMP+/− PARP+/+ and TRAMP+/− PARP-1−/− mice (20 weeks); magnification ×400. ( B ) Quantitative analysis of data from A, reveals a significant decrease in nuclear AR in prostate tissue from TRAMP+/−, PARP-1−/− mice compared with controls ( P

    Article Snippet: Animals (3–6 mice/group) were divided into the following experimental groups: (i) TRAMP−/−, PARP-1−/−; (ii) TRAMP−/−, PARP-1+/−; (iii) TRAMP−/−, PARP-1+/+; (iv) TRAMP+/−, PARP-1−/−; (v) TRAMP+/−, PARP-1+/−; (vi) TRAMP+/−, PARP-1+/+.

    Techniques: Activity Assay, Mouse Assay

    PARP-1 deficiency yields acquisition of EMT phenotype during prostate tumorigenesis. ( A ) Expression profile of E-cadherin and N-cadherin in prostate tissue from TRAMP+/− PARP-1+/+ and TRAMP+/− PARP-1−/− mice (28 weeks); magnification ×200. ( B ) Reveals immunoreactivity pattern for ZEB1 in prostate tumor sections from TRAMP+/−/PARP-1 +/+ and TRAMP+/−/PARP-1−/− mice. Magnification, ×200 and ×1000 (middle and right panels, respectively, ZEB-1 staining). Serial sections were stained with hematoxylin and eosin (left), revealing high grade, poorly differentiated prostate tumor from TRAMP+/− PARP−/− mice. ( C ) Shows western blot analysis of EMT regulators ZEB-1, E-cadherin, N-cadherin, β-catenin expression in prostate tissue from three different groups of TRAMP+/− PARP-1+/+ and TRAMP+/− PARP-1−/− mice (16 weeks). The three blots are representative of three independent groups of mice (groups I, II and III). ( D ) The barographs indicate the relative band intensity (from C) as determined by densitometry and expressed relative to glyceraldehyde 3-phosphate dehydrogenase (loading control). ( E ) Shows mRNA profile by reverse transcription–PCR analysis of E-cadherin, β-catenin , N-cadherin, ZEB-1 and Twist gene expression in TRAMP+/− PARP-1+/+ versus TRAMP+/− PARP-1−/− mice (16 weeks). Statistical significance (*) was determined at a value of P

    Journal: Carcinogenesis

    Article Title: PARP-1 regulates epithelial–mesenchymal transition (EMT) in prostate tumorigenesis

    doi: 10.1093/carcin/bgu183

    Figure Lengend Snippet: PARP-1 deficiency yields acquisition of EMT phenotype during prostate tumorigenesis. ( A ) Expression profile of E-cadherin and N-cadherin in prostate tissue from TRAMP+/− PARP-1+/+ and TRAMP+/− PARP-1−/− mice (28 weeks); magnification ×200. ( B ) Reveals immunoreactivity pattern for ZEB1 in prostate tumor sections from TRAMP+/−/PARP-1 +/+ and TRAMP+/−/PARP-1−/− mice. Magnification, ×200 and ×1000 (middle and right panels, respectively, ZEB-1 staining). Serial sections were stained with hematoxylin and eosin (left), revealing high grade, poorly differentiated prostate tumor from TRAMP+/− PARP−/− mice. ( C ) Shows western blot analysis of EMT regulators ZEB-1, E-cadherin, N-cadherin, β-catenin expression in prostate tissue from three different groups of TRAMP+/− PARP-1+/+ and TRAMP+/− PARP-1−/− mice (16 weeks). The three blots are representative of three independent groups of mice (groups I, II and III). ( D ) The barographs indicate the relative band intensity (from C) as determined by densitometry and expressed relative to glyceraldehyde 3-phosphate dehydrogenase (loading control). ( E ) Shows mRNA profile by reverse transcription–PCR analysis of E-cadherin, β-catenin , N-cadherin, ZEB-1 and Twist gene expression in TRAMP+/− PARP-1+/+ versus TRAMP+/− PARP-1−/− mice (16 weeks). Statistical significance (*) was determined at a value of P

    Article Snippet: Animals (3–6 mice/group) were divided into the following experimental groups: (i) TRAMP−/−, PARP-1−/−; (ii) TRAMP−/−, PARP-1+/−; (iii) TRAMP−/−, PARP-1+/+; (iv) TRAMP+/−, PARP-1−/−; (v) TRAMP+/−, PARP-1+/−; (vi) TRAMP+/−, PARP-1+/+.

    Techniques: Expressing, Mouse Assay, Staining, Western Blot, Polymerase Chain Reaction

    Mechanism of tumor suppressive function of CLDN7 in ccRCC. ( A ) Heatmap representation of differentially expressed genes identified by RNA-Seq between Caki-1 CLDN7 cells ( n = 3) and Caki-1 Control cells (n = 3). ( B ) Statistics of KEGG pathway enrichment. The y-axis corresponds to KEGG Pathway, and the x-axis shows the GeneRatio. The color of the dot represent adjusted p value (padj), and the size of the dot represents the number of differentially expressed genes mapped to the reference pathways. ( C ) Validation of differentially expressed genes by qRT-PCR. Comparison of mRNA expression of genes in pathways of cancer ( BCL2 , HIF1A , GLI-1 , ITGB-1 , p21 and AR ) and genes in EMT-related pathway ( TGFB1 , E-cadherin , N-cadherin , Vimentin and TWIST1 ) between Caki-1 CLDN7 cells and Caki-1 Control cells. All data are shown as means ± SD. ( D ) a . Western blot assay (left) and statistical analysis (right) of CLDN7, BCL2, cleaved-PARP1 and cleaved-Caspase 3 expression in Caki-1 and A498 cells, while overexpression of CLDN7 compared with control group. b . IHC assay of BCL2, cleaved-Caspase 3, Ki-67 and CLDN7 expression in xenografts formed by Caki-1 CLDN7 and Control cells. Scale bar, 100 μm. ( E ) a . Western blot assay and statistical analysis of E-cadherin, N-cadherin and Vimentin expression in CLDN7 overexpressed Caki-1 and A498 cells, comparing with control cells. b . IHC assay of E-cadherin, N-cadherin, TGFB1 and CLDN7 in xenografts formed by Caki-1 CLDN7 and Control cells. Scale bar, 100 μm. N.S, not significant. *p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Downregulation of CLDN7 due to promoter hypermethylation is associated with human clear cell renal cell carcinoma progression and poor prognosis

    doi: 10.1186/s13046-018-0924-y

    Figure Lengend Snippet: Mechanism of tumor suppressive function of CLDN7 in ccRCC. ( A ) Heatmap representation of differentially expressed genes identified by RNA-Seq between Caki-1 CLDN7 cells ( n = 3) and Caki-1 Control cells (n = 3). ( B ) Statistics of KEGG pathway enrichment. The y-axis corresponds to KEGG Pathway, and the x-axis shows the GeneRatio. The color of the dot represent adjusted p value (padj), and the size of the dot represents the number of differentially expressed genes mapped to the reference pathways. ( C ) Validation of differentially expressed genes by qRT-PCR. Comparison of mRNA expression of genes in pathways of cancer ( BCL2 , HIF1A , GLI-1 , ITGB-1 , p21 and AR ) and genes in EMT-related pathway ( TGFB1 , E-cadherin , N-cadherin , Vimentin and TWIST1 ) between Caki-1 CLDN7 cells and Caki-1 Control cells. All data are shown as means ± SD. ( D ) a . Western blot assay (left) and statistical analysis (right) of CLDN7, BCL2, cleaved-PARP1 and cleaved-Caspase 3 expression in Caki-1 and A498 cells, while overexpression of CLDN7 compared with control group. b . IHC assay of BCL2, cleaved-Caspase 3, Ki-67 and CLDN7 expression in xenografts formed by Caki-1 CLDN7 and Control cells. Scale bar, 100 μm. ( E ) a . Western blot assay and statistical analysis of E-cadherin, N-cadherin and Vimentin expression in CLDN7 overexpressed Caki-1 and A498 cells, comparing with control cells. b . IHC assay of E-cadherin, N-cadherin, TGFB1 and CLDN7 in xenografts formed by Caki-1 CLDN7 and Control cells. Scale bar, 100 μm. N.S, not significant. *p

    Article Snippet: Antibodies specific to CLDN7 (ab27487), BCL-2 (ab32124), PARP1 (ab32064), Caspase-3 (ab13847), E-cadherin (CDH1, ab76055), N-cadherin (CDH2, ab18203), Vimentin (ab92547) and TGFB1 (ab25121) were purchased from Abcam (Hong Kong, China).

    Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Expressing, Western Blot, Over Expression, Immunohistochemistry

    The effect of basic fibroblast growth factor (bFGF) on mitochondrial dysfunction-related proteins induced by hydroperoxide (TBHP) in H9C2 cells. (A) H9C2 cells were pre-treated with 50 ng/ml bFGF for 2 hrs, and then 100 μM TBHP was added for an additional 8 hrs. The cell lysates were analysed for the expression of Bax, Bcl-2, cleaved-PARP and cleaved-caspase-9 by western blotting. Bar diagram of Bax, Bcl-2, cleaved-PARP and cleaved-caspase-9 expression from three Western blot analyses. (B) Immunofluorescence results of the mitochondrial apoptotic marker cytochrome c in H9C2 cells. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: bFGF attenuates endoplasmic reticulum stress and mitochondrial injury on myocardial ischaemia/reperfusion via activation of PI3K/Akt/ERK1/2 pathway

    doi: 10.1111/jcmm.12346

    Figure Lengend Snippet: The effect of basic fibroblast growth factor (bFGF) on mitochondrial dysfunction-related proteins induced by hydroperoxide (TBHP) in H9C2 cells. (A) H9C2 cells were pre-treated with 50 ng/ml bFGF for 2 hrs, and then 100 μM TBHP was added for an additional 8 hrs. The cell lysates were analysed for the expression of Bax, Bcl-2, cleaved-PARP and cleaved-caspase-9 by western blotting. Bar diagram of Bax, Bcl-2, cleaved-PARP and cleaved-caspase-9 expression from three Western blot analyses. (B) Immunofluorescence results of the mitochondrial apoptotic marker cytochrome c in H9C2 cells. * P

    Article Snippet: Anti-Akt, p-Akt (Ser473), anti-ERK1/2, p-ERK1/2 (Thr202/Tyr204), anti-cleaved-caspase-3, cleaved-caspase-9, Bax, Bcl-2, cleaved-PARP, cytochrome c , anti-CHOP, cleaved-caspase-12, glucose-regulated protein (GRP-78), ATF-6 and GAPDH antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing, Western Blot, Immunofluorescence, Marker

    Immunofluorescent staining of endoplasmic reticulum (ER) stress and mitochondrial dysfunction-related proteins in the hearts of mice. (A) Immunofluorescent staining for GRP-78, CHOP, cleaved caspase-12 and cleaved-PARP in the hearts of control, ischaemia/reperfusion (I/R) mice and I/R mice treated with basic fibroblast growth factor (bFGF). (B) Analysis of the positive cells of the immunofluorescent results. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: bFGF attenuates endoplasmic reticulum stress and mitochondrial injury on myocardial ischaemia/reperfusion via activation of PI3K/Akt/ERK1/2 pathway

    doi: 10.1111/jcmm.12346

    Figure Lengend Snippet: Immunofluorescent staining of endoplasmic reticulum (ER) stress and mitochondrial dysfunction-related proteins in the hearts of mice. (A) Immunofluorescent staining for GRP-78, CHOP, cleaved caspase-12 and cleaved-PARP in the hearts of control, ischaemia/reperfusion (I/R) mice and I/R mice treated with basic fibroblast growth factor (bFGF). (B) Analysis of the positive cells of the immunofluorescent results. * P

    Article Snippet: Anti-Akt, p-Akt (Ser473), anti-ERK1/2, p-ERK1/2 (Thr202/Tyr204), anti-cleaved-caspase-3, cleaved-caspase-9, Bax, Bcl-2, cleaved-PARP, cytochrome c , anti-CHOP, cleaved-caspase-12, glucose-regulated protein (GRP-78), ATF-6 and GAPDH antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Staining, Mouse Assay

    Inhibition of the PI3K/Akt and ERK1/2 pathways partially attenuates the basic fibroblast growth factor (bFGF)-mediated reduction in the endoplasmic reticulum (ER) stress and mitochondrial dysfunction effects in H9C2 cells. H9C2 cells were pre-treated with 50 ng/ml bFGF with or without the specific inhibitors LY294002 (20 μM) and PD98059 (20 μM) for 2 hrs, and then 100 μM hydroperoxide (TBHP) was added for an additional 8 hrs. The cell lysates were analysed by western blotting to detect the expression of phospho-Akt, phospho-ERK and ERK, CHOP, GRP-78, ATF-6, caspase-12, Bax, Bcl-2, Cyt c, cleaved-PARP and cleaved-caspase-9. Bar diagram of the (B) phospho-Akt/Akt ratio, phospho-ERK/ERK ratio, (C) CHOP, GRP-78, ATF-6 and caspase-12, (D) Bax, Bcl-2, Cyt c, cleaved-PARP and cleaved-caspase-9 expression from three Western blot analyses. GAPDH was used as a protein loading control and for band density normalization. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: bFGF attenuates endoplasmic reticulum stress and mitochondrial injury on myocardial ischaemia/reperfusion via activation of PI3K/Akt/ERK1/2 pathway

    doi: 10.1111/jcmm.12346

    Figure Lengend Snippet: Inhibition of the PI3K/Akt and ERK1/2 pathways partially attenuates the basic fibroblast growth factor (bFGF)-mediated reduction in the endoplasmic reticulum (ER) stress and mitochondrial dysfunction effects in H9C2 cells. H9C2 cells were pre-treated with 50 ng/ml bFGF with or without the specific inhibitors LY294002 (20 μM) and PD98059 (20 μM) for 2 hrs, and then 100 μM hydroperoxide (TBHP) was added for an additional 8 hrs. The cell lysates were analysed by western blotting to detect the expression of phospho-Akt, phospho-ERK and ERK, CHOP, GRP-78, ATF-6, caspase-12, Bax, Bcl-2, Cyt c, cleaved-PARP and cleaved-caspase-9. Bar diagram of the (B) phospho-Akt/Akt ratio, phospho-ERK/ERK ratio, (C) CHOP, GRP-78, ATF-6 and caspase-12, (D) Bax, Bcl-2, Cyt c, cleaved-PARP and cleaved-caspase-9 expression from three Western blot analyses. GAPDH was used as a protein loading control and for band density normalization. * P

    Article Snippet: Anti-Akt, p-Akt (Ser473), anti-ERK1/2, p-ERK1/2 (Thr202/Tyr204), anti-cleaved-caspase-3, cleaved-caspase-9, Bax, Bcl-2, cleaved-PARP, cytochrome c , anti-CHOP, cleaved-caspase-12, glucose-regulated protein (GRP-78), ATF-6 and GAPDH antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Inhibition, Western Blot, Expressing

    Basic fibroblast growth factor (bFGF) reduces myocardial apoptosis and the caspase cascade pathway in the hearts of mice after myocardial ischaemia/reperfusion. (A) Representative terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) immunofluorescence of sections from the ischaemic area in the hearts of mice that received bFGF or vehicle. (B) The detection of endoplasmic reticulum (ER) stress-related and mitochondrial dysfunction-related apoptosis proteins was performed by western blotting. The protein expression levels of cleaved-PARP, caspase-3, caspase-9 and caspase-12 in the hearts of control, ischaemia/reperfusion (I/R) mice and I/R mice treated with bFGF. (C) The optical density analysis of cleaved-PARP, caspase-3, caspase-9 and caspase-12 in the heart. (D) The percentage of apoptosis was counted from three random 1 mm 2 areas. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: bFGF attenuates endoplasmic reticulum stress and mitochondrial injury on myocardial ischaemia/reperfusion via activation of PI3K/Akt/ERK1/2 pathway

    doi: 10.1111/jcmm.12346

    Figure Lengend Snippet: Basic fibroblast growth factor (bFGF) reduces myocardial apoptosis and the caspase cascade pathway in the hearts of mice after myocardial ischaemia/reperfusion. (A) Representative terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) immunofluorescence of sections from the ischaemic area in the hearts of mice that received bFGF or vehicle. (B) The detection of endoplasmic reticulum (ER) stress-related and mitochondrial dysfunction-related apoptosis proteins was performed by western blotting. The protein expression levels of cleaved-PARP, caspase-3, caspase-9 and caspase-12 in the hearts of control, ischaemia/reperfusion (I/R) mice and I/R mice treated with bFGF. (C) The optical density analysis of cleaved-PARP, caspase-3, caspase-9 and caspase-12 in the heart. (D) The percentage of apoptosis was counted from three random 1 mm 2 areas. * P

    Article Snippet: Anti-Akt, p-Akt (Ser473), anti-ERK1/2, p-ERK1/2 (Thr202/Tyr204), anti-cleaved-caspase-3, cleaved-caspase-9, Bax, Bcl-2, cleaved-PARP, cytochrome c , anti-CHOP, cleaved-caspase-12, glucose-regulated protein (GRP-78), ATF-6 and GAPDH antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Mouse Assay, TUNEL Assay, Immunofluorescence, Western Blot, Expressing

    3-AB inhibits mouse MOR mRNA expression in NS20Y cells and schematic model for PARP-1 in modulation of mouse MOR transcription. (A) Quantification of transcripts was performed by RT-PCR. Total RNA from NS20Y cells treated with 2 mM 3-AB was prepared and treated with DNase I. Primer pairs specific for the coding sequence of each gene were used for RT-PCR. PCR products were visualized in a 2% agarose gel. Lane 1: Molecular weight markers (M); lane 2: Control; lane 3: 3-AB-treated cells. (B) Quantitative analysis using ImageQuant 5.2 software. The MOR mRNA levels from Control and 3-AB-treated cells were normalized against β-actin levels. The values were obtained from triplicate data points. Changes in transcript levels for 3-AB-treated samples were compared to Control, which was assigned a value of 1.0. Bars indicate the range of standard error. (C) Schematic model for the role of PARP-1 in modulation of mouse MOR gene transcription. In neuronal cells, enzymatically active PARP-1 interacts strongly with the poly(C) sequence of the mouse MOR promoter and aids in the formation of tran-scriptionally inactive chromatin. Enzymatic inhibition of PARP-1 by 3-AB results in non-poly(ADP-ribosyl)ated PARP-1 and subsequently, an increase in the levels of MOR mRNA in mouse NS20Y cells.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Transcriptional regulation of mouse mu opioid receptor gene in neuronal cells by Poly(ADP-ribose) polymerase-1

    doi: 10.1111/j.1582-4934.2008.00259.x

    Figure Lengend Snippet: 3-AB inhibits mouse MOR mRNA expression in NS20Y cells and schematic model for PARP-1 in modulation of mouse MOR transcription. (A) Quantification of transcripts was performed by RT-PCR. Total RNA from NS20Y cells treated with 2 mM 3-AB was prepared and treated with DNase I. Primer pairs specific for the coding sequence of each gene were used for RT-PCR. PCR products were visualized in a 2% agarose gel. Lane 1: Molecular weight markers (M); lane 2: Control; lane 3: 3-AB-treated cells. (B) Quantitative analysis using ImageQuant 5.2 software. The MOR mRNA levels from Control and 3-AB-treated cells were normalized against β-actin levels. The values were obtained from triplicate data points. Changes in transcript levels for 3-AB-treated samples were compared to Control, which was assigned a value of 1.0. Bars indicate the range of standard error. (C) Schematic model for the role of PARP-1 in modulation of mouse MOR gene transcription. In neuronal cells, enzymatically active PARP-1 interacts strongly with the poly(C) sequence of the mouse MOR promoter and aids in the formation of tran-scriptionally inactive chromatin. Enzymatic inhibition of PARP-1 by 3-AB results in non-poly(ADP-ribosyl)ated PARP-1 and subsequently, an increase in the levels of MOR mRNA in mouse NS20Y cells.

    Article Snippet: One microgram of anti-PARP-1 produced a minor supershifted band (arrow), while 2 μg of anti-PARP-1 produced a supershifted band (arrow) with concomitant reduction in the intensity of the complex band (asterisk).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Sequencing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Molecular Weight, Software, Inhibition

    Analysis of mouse MOR gene regulation by PARP-1 in vivo using siRNA. PARP-1 siRNA increases MOR transcription in NS20Y cells. (A) NS20Y cells were trans-fected with either scrambled siRNA (Scb) or PARP-1 siRNA (siPARP-1). Whole-cell extracts were made after incubation with the siRNAs for 48 hrs. Immunoblot analyses for PARP-1 and β-actin were performed. This figure is a representative of three separate experiments. (B) Quantification of MOR transcripts was performed by RT-PCR. Total RNA from NS20Y cells was prepared and treated with DNase I, and primer pairs specific for the coding sequence of each gene were used for RT-PCR. (C) Quantitative analysis using ImageQuant 5.2 software. The MOR mRNA levels from Control, scrambled (Scb) or siRNA-treated (siPARP-1) cells were normalized against β-actin levels. The values were obtained from triplicate data points and changes in transcript levels for Scb or siPARP-1-treated samples were compared to Control, which was assigned a value of 1.0. Bars indicate the range of standard error.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Transcriptional regulation of mouse mu opioid receptor gene in neuronal cells by Poly(ADP-ribose) polymerase-1

    doi: 10.1111/j.1582-4934.2008.00259.x

    Figure Lengend Snippet: Analysis of mouse MOR gene regulation by PARP-1 in vivo using siRNA. PARP-1 siRNA increases MOR transcription in NS20Y cells. (A) NS20Y cells were trans-fected with either scrambled siRNA (Scb) or PARP-1 siRNA (siPARP-1). Whole-cell extracts were made after incubation with the siRNAs for 48 hrs. Immunoblot analyses for PARP-1 and β-actin were performed. This figure is a representative of three separate experiments. (B) Quantification of MOR transcripts was performed by RT-PCR. Total RNA from NS20Y cells was prepared and treated with DNase I, and primer pairs specific for the coding sequence of each gene were used for RT-PCR. (C) Quantitative analysis using ImageQuant 5.2 software. The MOR mRNA levels from Control, scrambled (Scb) or siRNA-treated (siPARP-1) cells were normalized against β-actin levels. The values were obtained from triplicate data points and changes in transcript levels for Scb or siPARP-1-treated samples were compared to Control, which was assigned a value of 1.0. Bars indicate the range of standard error.

    Article Snippet: One microgram of anti-PARP-1 produced a minor supershifted band (arrow), while 2 μg of anti-PARP-1 produced a supershifted band (arrow) with concomitant reduction in the intensity of the complex band (asterisk).

    Techniques: In Vivo, Incubation, Reverse Transcription Polymerase Chain Reaction, Sequencing, Software

    PARP-1 represses the proximal promoter of the mouse MOR gene. (A) Schematic representations of the mouse MOR proximal promoter region (the PARP-1 -binding motif is underlined), the pGL450 (wild-type) promoter construct and the pGL450mut construct (containing a mutated PARP-1 binding site). The ‘X’ in the filled ovals indicates the mutation, which includes the PARP-1 binding site and its flanking sequence. Nucleotide +1 corresponds to the translation start site (ATG). (B, C) Neuronal NS20Y cells endogenously expressing the MOR gene were co-transfected with 2 pg of the PARP-1 constructs and 1 pg of the MOR-promoter luciferase-reporter constructs, pGL450 and pGL450mut. The activities of the luciferase reporter were expressed as n-fold relative to the activity of each corresponding luciferase reporter transfected with vector alone, which was assigned an activity value of 1.0. Transfection efficiencies were normalized by β-galactosidase activity. The data shown are the mean and standard errors of three independent experiments with at least two different plasmid preparations.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Transcriptional regulation of mouse mu opioid receptor gene in neuronal cells by Poly(ADP-ribose) polymerase-1

    doi: 10.1111/j.1582-4934.2008.00259.x

    Figure Lengend Snippet: PARP-1 represses the proximal promoter of the mouse MOR gene. (A) Schematic representations of the mouse MOR proximal promoter region (the PARP-1 -binding motif is underlined), the pGL450 (wild-type) promoter construct and the pGL450mut construct (containing a mutated PARP-1 binding site). The ‘X’ in the filled ovals indicates the mutation, which includes the PARP-1 binding site and its flanking sequence. Nucleotide +1 corresponds to the translation start site (ATG). (B, C) Neuronal NS20Y cells endogenously expressing the MOR gene were co-transfected with 2 pg of the PARP-1 constructs and 1 pg of the MOR-promoter luciferase-reporter constructs, pGL450 and pGL450mut. The activities of the luciferase reporter were expressed as n-fold relative to the activity of each corresponding luciferase reporter transfected with vector alone, which was assigned an activity value of 1.0. Transfection efficiencies were normalized by β-galactosidase activity. The data shown are the mean and standard errors of three independent experiments with at least two different plasmid preparations.

    Article Snippet: One microgram of anti-PARP-1 produced a minor supershifted band (arrow), while 2 μg of anti-PARP-1 produced a supershifted band (arrow) with concomitant reduction in the intensity of the complex band (asterisk).

    Techniques: Binding Assay, Construct, Mutagenesis, Sequencing, Expressing, Transfection, Luciferase, Activity Assay, Plasmid Preparation

    EMSA analysis of the PARP-1-binding motif using mutant oligonucleotide sequences and ChIP assay. (A) Representation of the double-stranded oligonucleotide sequence (NS) and mutant oligonucleotide sequences (M1–M8). (B) EMSAs were performed using unlabelled poly(C) sequence (NS; lane 2) or unlabelled poly(C) mutated sequences (M1–M8; lanes 3–10) as competitors for recombinant PARP-1 protein binding to a labelled poly(C) sequence. Lane 1: Negative control (no unlabelled poly(C) sequence). The PARP-1-poly(C) sequence complex is indicated by an arrow. (C) The PARP-1-binding motif of the poly(C) sequence (NS). (D) ChIP analysis by real-time qPCR for PARP-1 binding interaction with the MOR promoter poly(C) sequence. Interactions were examined by ChIP assay with anti-PARP antibody and nonspecific antibody (anti-gal4). Precipitated DNAs were amplified using mouse MOR and β-actin (negative control) primers.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Transcriptional regulation of mouse mu opioid receptor gene in neuronal cells by Poly(ADP-ribose) polymerase-1

    doi: 10.1111/j.1582-4934.2008.00259.x

    Figure Lengend Snippet: EMSA analysis of the PARP-1-binding motif using mutant oligonucleotide sequences and ChIP assay. (A) Representation of the double-stranded oligonucleotide sequence (NS) and mutant oligonucleotide sequences (M1–M8). (B) EMSAs were performed using unlabelled poly(C) sequence (NS; lane 2) or unlabelled poly(C) mutated sequences (M1–M8; lanes 3–10) as competitors for recombinant PARP-1 protein binding to a labelled poly(C) sequence. Lane 1: Negative control (no unlabelled poly(C) sequence). The PARP-1-poly(C) sequence complex is indicated by an arrow. (C) The PARP-1-binding motif of the poly(C) sequence (NS). (D) ChIP analysis by real-time qPCR for PARP-1 binding interaction with the MOR promoter poly(C) sequence. Interactions were examined by ChIP assay with anti-PARP antibody and nonspecific antibody (anti-gal4). Precipitated DNAs were amplified using mouse MOR and β-actin (negative control) primers.

    Article Snippet: One microgram of anti-PARP-1 produced a minor supershifted band (arrow), while 2 μg of anti-PARP-1 produced a supershifted band (arrow) with concomitant reduction in the intensity of the complex band (asterisk).

    Techniques: Binding Assay, Mutagenesis, Chromatin Immunoprecipitation, Sequencing, Recombinant, Protein Binding, Negative Control, Real-time Polymerase Chain Reaction, Amplification

    Auto-poly(ADP-ribosyl)ation of PARP-1 in vitvo w’froand EMSA of poly(C)-binding sequence with recombinant PARP-1 and purified proteins. (A) The MOR poly(C) sequence (NS). (B) Auto-poly(ADP-ribosyl)ation of PARP-1 in vitro. Recombinant PARP-1 was incubated in the absence or presence of 10 mM 3-AB for 20 min. PARP-1 and poly(ADP-ribosyl)ated PARP-1 were detected using anti-PARP-1 and anti-poly(ADP-ribose) (anti-PAR). Lane 1: control (nonenzymatic reaction without NAD + ); lane 2: enzymatic reaction with NAD + ; lane 3: inhibited enzymatic reaction with NAD + and 3-AB. (C) EMSAs were performed using the labelled poly(C) sequence (NS) and unpoly(ADP-ribosyl)ated or poly(ADP-ribosyl)ated PARP-1. Lane 1: probe alone; lane 2: unpoly(ADP-ribosyl)ated PARP-1; lane 3: poly(ADP-ribosyl)ated PARP-1; lane 4: poly(ADP-ribosyl)ation of PARP-1 inhibited by 3-AB; lane 5: unpoly(ADP-ribosyl)ated PARP-1 in the presence of competitor; lane 6: poly(ADP-ribosyl)ation of PARP-1 in the presence of competitor; lane 7: poly(ADP-ribosyl)ation of PARP-1 in the presence of competitor and 3-AB inhibitor. The PARP-1-poly(C) sequence complex is indicated by an arrow. (D) Coomassie-stained gel of poly(C)-binding proteins purified from NS20Y nuclear extracts and western blot analysis of purified poly(C)-binding proteins probed with anti-PARP-1 and anti-PAR antibodies. Arrows indicate PARP-1, poly(ADP-ribosyl)ated PARP-1 and poly(ADP-ribosyl)ated proteins. (E) EMSA of purified poly(C)-binding proteins using anti-PARP and anti-PAR antibody. EMSAs were performed using the 32p-labelled MOR poly(C) sequence (NS) as a probe with purified poly(C)-binding proteins. Lane 1: Self-competitor without antibody; lane 2: EMSA reaction without antibody; lane 3: EMSA with anti-PARP antibody (1 μg); lane 4: EMSA with anti-PARP antibody (2 μg); lane 5: EMSA with anti-PAR antibody. Supershifted bands of PARP antibody and PAR antibody are indicated by arrows.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Transcriptional regulation of mouse mu opioid receptor gene in neuronal cells by Poly(ADP-ribose) polymerase-1

    doi: 10.1111/j.1582-4934.2008.00259.x

    Figure Lengend Snippet: Auto-poly(ADP-ribosyl)ation of PARP-1 in vitvo w’froand EMSA of poly(C)-binding sequence with recombinant PARP-1 and purified proteins. (A) The MOR poly(C) sequence (NS). (B) Auto-poly(ADP-ribosyl)ation of PARP-1 in vitro. Recombinant PARP-1 was incubated in the absence or presence of 10 mM 3-AB for 20 min. PARP-1 and poly(ADP-ribosyl)ated PARP-1 were detected using anti-PARP-1 and anti-poly(ADP-ribose) (anti-PAR). Lane 1: control (nonenzymatic reaction without NAD + ); lane 2: enzymatic reaction with NAD + ; lane 3: inhibited enzymatic reaction with NAD + and 3-AB. (C) EMSAs were performed using the labelled poly(C) sequence (NS) and unpoly(ADP-ribosyl)ated or poly(ADP-ribosyl)ated PARP-1. Lane 1: probe alone; lane 2: unpoly(ADP-ribosyl)ated PARP-1; lane 3: poly(ADP-ribosyl)ated PARP-1; lane 4: poly(ADP-ribosyl)ation of PARP-1 inhibited by 3-AB; lane 5: unpoly(ADP-ribosyl)ated PARP-1 in the presence of competitor; lane 6: poly(ADP-ribosyl)ation of PARP-1 in the presence of competitor; lane 7: poly(ADP-ribosyl)ation of PARP-1 in the presence of competitor and 3-AB inhibitor. The PARP-1-poly(C) sequence complex is indicated by an arrow. (D) Coomassie-stained gel of poly(C)-binding proteins purified from NS20Y nuclear extracts and western blot analysis of purified poly(C)-binding proteins probed with anti-PARP-1 and anti-PAR antibodies. Arrows indicate PARP-1, poly(ADP-ribosyl)ated PARP-1 and poly(ADP-ribosyl)ated proteins. (E) EMSA of purified poly(C)-binding proteins using anti-PARP and anti-PAR antibody. EMSAs were performed using the 32p-labelled MOR poly(C) sequence (NS) as a probe with purified poly(C)-binding proteins. Lane 1: Self-competitor without antibody; lane 2: EMSA reaction without antibody; lane 3: EMSA with anti-PARP antibody (1 μg); lane 4: EMSA with anti-PARP antibody (2 μg); lane 5: EMSA with anti-PAR antibody. Supershifted bands of PARP antibody and PAR antibody are indicated by arrows.

    Article Snippet: One microgram of anti-PARP-1 produced a minor supershifted band (arrow), while 2 μg of anti-PARP-1 produced a supershifted band (arrow) with concomitant reduction in the intensity of the complex band (asterisk).

    Techniques: Binding Assay, Sequencing, Recombinant, Purification, In Vitro, Incubation, Staining, Western Blot

    Identification of PARP-1 as a poly(C)-binding protein. Mascot results of the mass spectrometry identification of the 124-kD protein band. The value with the highest score (111) identifies the protein as PARP-1.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Transcriptional regulation of mouse mu opioid receptor gene in neuronal cells by Poly(ADP-ribose) polymerase-1

    doi: 10.1111/j.1582-4934.2008.00259.x

    Figure Lengend Snippet: Identification of PARP-1 as a poly(C)-binding protein. Mascot results of the mass spectrometry identification of the 124-kD protein band. The value with the highest score (111) identifies the protein as PARP-1.

    Article Snippet: One microgram of anti-PARP-1 produced a minor supershifted band (arrow), while 2 μg of anti-PARP-1 produced a supershifted band (arrow) with concomitant reduction in the intensity of the complex band (asterisk).

    Techniques: Binding Assay, Mass Spectrometry