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  • 94
    Millipore parp
    E2F1 localizes to mitochondria in Saos‐2 cells, where it promotes apoptosis Apoptotic signal induces E2F1 stabilization. Saos‐2 cells were treated for 16 h by 50 μM etoposide or not (untreated) before Western blot analysis of E2F1 expression and <t>PARP1</t> cleavage. Etoposide induces apoptosis in E2F1‐dependent manner. Saos‐2 cells were transfected with control or E2F1 siRNA for 24 h and treated as in (A) before cell death analysis by trypan blue staining. Western blot controlling the E2F1 siRNA extinction is inserted. E2F1 constitutively localizes to mitochondria. Saos‐2 cells were fractionated, and equal amounts of total lysate versus heavy membrane fraction (HM fraction) or mitochondrial‐enriched fraction (Mito fraction) were analyzed by Western blot analysis for E2F1 and BCL‐xL expression. <t>KTN,</t> LAMIN A/C, and COX IV serve as markers of endoplasmic reticulum, nuclei, and mitochondria, respectively. Data shown are representative of at least three independent experiments. Ectopic E2F1 expression triggers apoptosis. Saos‐2 cells were transfected with the indicated E2F1 expression vectors and treated or not with etoposide (50 μM) for an additional 48 h. Apoptosis was evaluated by Annexin V‐APC staining among GFP‐positive cells using flow cytometry analysis. E2F1 triggers MOMP. MDA‐MB231 cells stably expressing OMI‐mCherry were transfected with the indicated expression vectors. 48 h post‐transfection, MOMP was quantified by determining the mCherry low fluorescence cell percentage among GFP‐positive cells using flow cytometry analysis. Data information: * P
    Parp, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 327 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Source BioScience plc length artd1 parp1
    Comparison of WIKI4 binding to tankyrase 2 with other tankyrase selective inhibitors and with <t>ARTD1-3</t> structures. WIKI4 - TNKS2 protein structure is colored in turquoise and WIKI4 is colored in lilac. a) Comparison of the WIKI4 binding sites in TNKS2, <t>ARTD1</t> (pink) (pdb accession code 3GJW), ARTD2 (green) (pdb accession code 3KCZ), and ARTD3 (red) (pdb accession code 3FHB). ARD, ARTD regulatory domain. b) Comparison of the binding of WIKI4 and IWR-1. Hydrogen bonds for WIKI4 and IWR-1 are shown in black and gray dotted lines, respectively. IWR-1 - TNKS2 protein structure is colored in pink and IWR-1 is colored in orange. c) Comparison of the binding of WIKI4 and G007-LK. Hydrogen bonds for WIKI4 and G007-LK are shown in black and gray dotted lines, respectively. G007-LK - TNKS2 protein structure is colored in pink and G007-LK is colored in orange.
    Length Artd1 Parp1, supplied by Source BioScience plc, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    ABclonal parp1
    Apoptosis related proteins were detected in EOMA cells after treated with DHTS and propranolol. (A,B) DHTS induced <t>PARP,</t> Aif, Caspase9, Caspase3, Bax and Cyts3 in low concentration more than in high concentration, while induced FADD and Caspase 8 more in relatively high concentration. (C,D) Propranolol induced PARP, Aif, Caspase9, Caspase3, Bax and Cyts3 in high concentration more than in low concentration, while induced FADD and Caspase 8 more in relatively low concentration.
    Parp1, supplied by ABclonal, used in various techniques. Bioz Stars score: 93/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc parp antibody
    ( a ) CLSM images indicating mitochondrial membrane potential change by JC-1 staining of U87-MG cells incubated with 40 μM AuCs for 3, 6, 12 h; ( b ) Apoptosis analysis of U87-MG cells by flow cytometry after incubation 20, 40, 60 μM AuCs for 48 h; ( c ) Western blot analysis of <t>caspase-3,</t> caspase-7, <t>PARP,</t> and their cleaved forms after cells were treated by 20, 40, and 60 μM AuCs for 48 h.
    Parp Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 498 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/parp antibody/product/Cell Signaling Technology Inc
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    88
    Santa Cruz Biotechnology α parp1
    ( a ) CLSM images indicating mitochondrial membrane potential change by JC-1 staining of U87-MG cells incubated with 40 μM AuCs for 3, 6, 12 h; ( b ) Apoptosis analysis of U87-MG cells by flow cytometry after incubation 20, 40, 60 μM AuCs for 48 h; ( c ) Western blot analysis of <t>caspase-3,</t> caspase-7, <t>PARP,</t> and their cleaved forms after cells were treated by 20, 40, and 60 μM AuCs for 48 h.
    α Parp1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti parp1
    Detection of apoptosis in retinas of rbpr2 fs-muz99 mutants. ( A ) Wild-type (WT) and rbpr2 mutant ( rbpr2 fs-muz99 ) zebrafish retinas at 5.5 dpf were stained with TUNEL reagent. The TUNEL positive cells/apoptotic nuclei stain green. ( B ) Quantification of TUNEL positive cells. ( C ) Determination of rod and cone opsin levels in WT and mutant eyes (dissected heads; n = 12 each genotype) by Western blot analysis at 8 dpf. ( D ) Total protein from WT and mutant eyes (dissected heads; n = 12 each genotype), at the 5.5 and 8 dpf time-points, were pooled and subjected to Western blot analysis using <t>PARP1</t> antibody. Cleaved PARP1 products in rbpr2 fs-muz99 mutant eyes indicate apoptosis. TUNEL assays and Western blot experiments were repeated thrice. For Figure 9 B, statistical analysis of data using the Mann–Whitney U test showed a p
    Anti Parp1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 307 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore human parp1 enzyme
    PARylation of Histone H3 decreases EZH2-mediated histone methylation (A) Schematic of experimental approach that couples histone PARylation and methylation in vitro . First, histone H3 was incubated with <t>PARP1</t> in the presence or absence of NAD+ and olaparib to allow for PARylation. After 60 minutes, the reaction was blocked by addition of olaparib, PARP1 was removed by immunoprecipitation and the remaining histone H3 was either assessed for PARylation by PAR-resin pulldown or incubated with EZH2/PRC2 and SAM to allow H3-K27 methylation in vitro . After 30 minutes, the histone methyltransferase reaction was blocked and H3K27me3 levels were determined by different approaches. (B) Histone H3 PARylation decreases subsequent H3-K27 methylation. Histone H3 proteins treated as in A) were analyzed by western blot using an anti-H3K27me3 antibody and an anti-histone H3 antibody as a control. The signal intensity of H3K27me3 relative to H3 was measured using ImageJ software and normalized to the signal from unmodified histone H3 (H3 incubated with PARP1 in the absence of NAD+, lane 1). PARP1 activity was confirmed by western blot using an anti-PAR antibody. The western blot is representative of three independent experiments. (C) PARylation reduces histone methylation in vitro . Histone H3 samples treated as in A) were used to determine EZH2 activity toward unmodified and PARylated histone H3 by measuring H3K27me3 levels using an ELISA kit. H3K27me3 levels from unmodified histone H3 were set as 100% EZH2 activity. N=3, mean ± SD. (D) In vitro PARylation of histone H3. Histone H3 proteins treated as in A) were immunoprecipitated using the PAR-affinity resin and PARylation of H3 was confirmed by western blot using an anti-H3 antibody. PAR-resin specificity and PARylation levels were determined by western blot analysis of purified proteins with an anti-PAR antibody. The smear observed in lane 2 indicated PARylation. H. (E) PARP1 Immunoprecipitation. PARP1 removal after PARylation of histone H3 treated as above was confirmed by western blot analysis of proteins immunoprecipitated with an anti-PARP1 antibody.
    Human Parp1 Enzyme, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    The Jackson Laboratory parp1
    RT-PCR of HDACI expression in THP-1 cells treated with LPS in the presence or absence of DIQ (300 μmol/L), a <t>PARP1</t> inhibitor. THP-1 cells were treated with LPS (10 μg/mL) for 4 or 8 h. Error bars indicate mean ± SEM.
    Parp1, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 92/100, based on 152 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Trevigen parp1
    HCT116 <t>PARP1</t> -/- cells are radiosensitive. (A) HCT116 PARP1 -/- cells (clones C2 and C4) and control PARP1-proficient HCT116 EV cells were exposed to IR (5 Gy) and counted after 48 hours. Bars represent the average and standard deviation of triplicates. Data is representative of two independent experiments. (B-C) Clonogenic assay after exposure to the indicated doses of IR. The surviving fraction is plotted in (B) and representative plates are shown in (C). Data is representative of two independent experiments. (D) Cell cycle profile after IR (5 Gy) at the indicated timepoints (hours after radiation). The percentage of cells with 2N and 4N DNA content is indicated. Data is representative of three independent experiments. (E-G) Quantification of irradiation-induced foci (IRIF). Cells were exposed to IR (2 Gy) and the number of foci per nucleus quantified by indirect immunofluorescence with antibodies to γ -H2AX and 53BP1. Histograms on (E) show the distribution of γ-H2AX foci per nucleus at baseline and 6 and 12 hours after IR. The percentage of cells with more than 10 γ-H2AX foci at the same timepoints is shown in F. Bars represent the average and standard deviation of three fields, N = 100 cells/field. (H) Cells extracts were harvested at the indicated timepoints after IR (4 Gy) and probed with antibodies to phospho-KAP1 (S824) and, as a loading control, GAPDH.
    Parp1, supplied by Trevigen, used in various techniques. Bioz Stars score: 92/100, based on 315 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    E2F1 localizes to mitochondria in Saos‐2 cells, where it promotes apoptosis Apoptotic signal induces E2F1 stabilization. Saos‐2 cells were treated for 16 h by 50 μM etoposide or not (untreated) before Western blot analysis of E2F1 expression and PARP1 cleavage. Etoposide induces apoptosis in E2F1‐dependent manner. Saos‐2 cells were transfected with control or E2F1 siRNA for 24 h and treated as in (A) before cell death analysis by trypan blue staining. Western blot controlling the E2F1 siRNA extinction is inserted. E2F1 constitutively localizes to mitochondria. Saos‐2 cells were fractionated, and equal amounts of total lysate versus heavy membrane fraction (HM fraction) or mitochondrial‐enriched fraction (Mito fraction) were analyzed by Western blot analysis for E2F1 and BCL‐xL expression. KTN, LAMIN A/C, and COX IV serve as markers of endoplasmic reticulum, nuclei, and mitochondria, respectively. Data shown are representative of at least three independent experiments. Ectopic E2F1 expression triggers apoptosis. Saos‐2 cells were transfected with the indicated E2F1 expression vectors and treated or not with etoposide (50 μM) for an additional 48 h. Apoptosis was evaluated by Annexin V‐APC staining among GFP‐positive cells using flow cytometry analysis. E2F1 triggers MOMP. MDA‐MB231 cells stably expressing OMI‐mCherry were transfected with the indicated expression vectors. 48 h post‐transfection, MOMP was quantified by determining the mCherry low fluorescence cell percentage among GFP‐positive cells using flow cytometry analysis. Data information: * P

    Journal: EMBO Reports

    Article Title: E2F1 interacts with BCL‐ xL and regulates its subcellular localization dynamics to trigger cell death

    doi: 10.15252/embr.201744046

    Figure Lengend Snippet: E2F1 localizes to mitochondria in Saos‐2 cells, where it promotes apoptosis Apoptotic signal induces E2F1 stabilization. Saos‐2 cells were treated for 16 h by 50 μM etoposide or not (untreated) before Western blot analysis of E2F1 expression and PARP1 cleavage. Etoposide induces apoptosis in E2F1‐dependent manner. Saos‐2 cells were transfected with control or E2F1 siRNA for 24 h and treated as in (A) before cell death analysis by trypan blue staining. Western blot controlling the E2F1 siRNA extinction is inserted. E2F1 constitutively localizes to mitochondria. Saos‐2 cells were fractionated, and equal amounts of total lysate versus heavy membrane fraction (HM fraction) or mitochondrial‐enriched fraction (Mito fraction) were analyzed by Western blot analysis for E2F1 and BCL‐xL expression. KTN, LAMIN A/C, and COX IV serve as markers of endoplasmic reticulum, nuclei, and mitochondria, respectively. Data shown are representative of at least three independent experiments. Ectopic E2F1 expression triggers apoptosis. Saos‐2 cells were transfected with the indicated E2F1 expression vectors and treated or not with etoposide (50 μM) for an additional 48 h. Apoptosis was evaluated by Annexin V‐APC staining among GFP‐positive cells using flow cytometry analysis. E2F1 triggers MOMP. MDA‐MB231 cells stably expressing OMI‐mCherry were transfected with the indicated expression vectors. 48 h post‐transfection, MOMP was quantified by determining the mCherry low fluorescence cell percentage among GFP‐positive cells using flow cytometry analysis. Data information: * P

    Article Snippet: The following antibodies were used: ACTIN (MAB1501R) and BIM (AB17003) from Millipore, E2F1 (3742), COXIV (4850), PUMA (4976), BID (2002S), and BAK (3814) from Cell Signaling, BAX (A3533) and BCL‐2 (M0887) from Dako, BCL‐xL ([E18] Ab32370) and GFP (Ab290) from Abcam, MCL‐1 (sc‐819), LAMIN A/C (sc‐376248), and KTN (sc‐33562) from Santa Cruz, and PARP (#AM30) from Calbiochem.

    Techniques: Western Blot, Expressing, Transfection, Staining, Flow Cytometry, Cytometry, Multiple Displacement Amplification, Stable Transfection, Fluorescence

    Comparison of WIKI4 binding to tankyrase 2 with other tankyrase selective inhibitors and with ARTD1-3 structures. WIKI4 - TNKS2 protein structure is colored in turquoise and WIKI4 is colored in lilac. a) Comparison of the WIKI4 binding sites in TNKS2, ARTD1 (pink) (pdb accession code 3GJW), ARTD2 (green) (pdb accession code 3KCZ), and ARTD3 (red) (pdb accession code 3FHB). ARD, ARTD regulatory domain. b) Comparison of the binding of WIKI4 and IWR-1. Hydrogen bonds for WIKI4 and IWR-1 are shown in black and gray dotted lines, respectively. IWR-1 - TNKS2 protein structure is colored in pink and IWR-1 is colored in orange. c) Comparison of the binding of WIKI4 and G007-LK. Hydrogen bonds for WIKI4 and G007-LK are shown in black and gray dotted lines, respectively. G007-LK - TNKS2 protein structure is colored in pink and G007-LK is colored in orange.

    Journal: PLoS ONE

    Article Title: Structural Basis and Selectivity of Tankyrase Inhibition by a Wnt Signaling Inhibitor WIKI4

    doi: 10.1371/journal.pone.0065404

    Figure Lengend Snippet: Comparison of WIKI4 binding to tankyrase 2 with other tankyrase selective inhibitors and with ARTD1-3 structures. WIKI4 - TNKS2 protein structure is colored in turquoise and WIKI4 is colored in lilac. a) Comparison of the WIKI4 binding sites in TNKS2, ARTD1 (pink) (pdb accession code 3GJW), ARTD2 (green) (pdb accession code 3KCZ), and ARTD3 (red) (pdb accession code 3FHB). ARD, ARTD regulatory domain. b) Comparison of the binding of WIKI4 and IWR-1. Hydrogen bonds for WIKI4 and IWR-1 are shown in black and gray dotted lines, respectively. IWR-1 - TNKS2 protein structure is colored in pink and IWR-1 is colored in orange. c) Comparison of the binding of WIKI4 and G007-LK. Hydrogen bonds for WIKI4 and G007-LK are shown in black and gray dotted lines, respectively. G007-LK - TNKS2 protein structure is colored in pink and G007-LK is colored in orange.

    Article Snippet: Cloning, Protein Expression and Protein Purification cDNA for full length ARTD1/PARP1 was purchased from Source Bioscience and the codon optimized full length ARTD2/PARP2 was purchased from Genescript.

    Techniques: Binding Assay

    Apoptosis related proteins were detected in EOMA cells after treated with DHTS and propranolol. (A,B) DHTS induced PARP, Aif, Caspase9, Caspase3, Bax and Cyts3 in low concentration more than in high concentration, while induced FADD and Caspase 8 more in relatively high concentration. (C,D) Propranolol induced PARP, Aif, Caspase9, Caspase3, Bax and Cyts3 in high concentration more than in low concentration, while induced FADD and Caspase 8 more in relatively low concentration.

    Journal: Frontiers in Pharmacology

    Article Title: 15, 16-Dihydrotanshinone I Inhibits Hemangiomas through Inducing Pro-apoptotic and Anti-angiogenic Mechanisms in Vitro and in Vivo

    doi: 10.3389/fphar.2018.00025

    Figure Lengend Snippet: Apoptosis related proteins were detected in EOMA cells after treated with DHTS and propranolol. (A,B) DHTS induced PARP, Aif, Caspase9, Caspase3, Bax and Cyts3 in low concentration more than in high concentration, while induced FADD and Caspase 8 more in relatively high concentration. (C,D) Propranolol induced PARP, Aif, Caspase9, Caspase3, Bax and Cyts3 in high concentration more than in low concentration, while induced FADD and Caspase 8 more in relatively low concentration.

    Article Snippet: Antibodies to Bax (1:1000, A0207), Aif (1:1000, A2568), Parp (1:1000, A0942), Caspase3 (1:1000, A0214), Caspase8 (1:1000, A0215), Caspase9 (1:1000, A11451), Cyst3 (1:1000, A1561), GAPDH (1:1000, AC001) and FADD (1:1000, A5819) were from Abclonal.

    Techniques: Concentration Assay

    ( a ) CLSM images indicating mitochondrial membrane potential change by JC-1 staining of U87-MG cells incubated with 40 μM AuCs for 3, 6, 12 h; ( b ) Apoptosis analysis of U87-MG cells by flow cytometry after incubation 20, 40, 60 μM AuCs for 48 h; ( c ) Western blot analysis of caspase-3, caspase-7, PARP, and their cleaved forms after cells were treated by 20, 40, and 60 μM AuCs for 48 h.

    Journal: Nanomaterials

    Article Title: Peptide-Templated Gold Clusters as Enzyme-Like Catalyst Boost Intracellular Oxidative Pressure and Induce Tumor-Specific Cell Apoptosis

    doi: 10.3390/nano8121040

    Figure Lengend Snippet: ( a ) CLSM images indicating mitochondrial membrane potential change by JC-1 staining of U87-MG cells incubated with 40 μM AuCs for 3, 6, 12 h; ( b ) Apoptosis analysis of U87-MG cells by flow cytometry after incubation 20, 40, 60 μM AuCs for 48 h; ( c ) Western blot analysis of caspase-3, caspase-7, PARP, and their cleaved forms after cells were treated by 20, 40, and 60 μM AuCs for 48 h.

    Article Snippet: Apoptosis Detection Kit (Annexin V-FITC/PI) and cell counting kit-8 (CCK-8) were acquired from Dojindo Laboratories (Kumamoto, Japan). β-actin antibody, PARP antibody, anti-rabbit IgG-HRP and caspase-3 antibody were purchased by Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Confocal Laser Scanning Microscopy, Staining, Incubation, Flow Cytometry, Cytometry, Western Blot

    PARP1 and CCL2 crosstalk regulate invasiveness in breast cancer. ( A ) Left: Recombinant human CCL2 (rhCCL2) can increase PAR levels in MB-231 cells. MB-231 cells were serum-starved overnight and treated with 50 ng/mL rhCCL2 for 1 h. Western blot showing total PAR level upon treatment. Right: Immunofluorescence images showing PAR (green) in cells after rhCCL2 treatment. An enlarged image in Figure S8 . Merged image with DNA (DAPI) shown on top. Scale bar 20 µm. ( B ) PARP1 inhibitor treatment reduces phosphorylated P44/42 (ERK1/2) levels in MB-231cells. Western blot showing Phospho-p44/42 ERK1/2 (Thr202/Tyr204) and total ERK1/2 after overnight PJ34 treatment followed by 45 min of TNFα. ( C ) Left: Heat map showing transcriptome analysis from MB-231 cells for rhCCL2 (1 h treatment as in A ) and PJ34 (10 µM Overnight treatment) treated groups. The KEGG MAP kinase pathway genes are identified. Red color indicates upregulation with green, showing the downregulation of mRNA transcription. Right: Ingenuity pathway analysis from rhCCL2 (Left column) and PJ34 treated (right column). Enlarged images of ( C ) are in Figure S8 . ( D ) Wound healing assay with simultaneous rhCCL2 and PJ34 treatment on MB-231 cells. Near confluent cells were pretreated with PJ34 before rhCCL2 was added at the dose of 50 ng/mL after wound creation. Treatment was continued for 12 h. The graph on the right shows % wound closure with respect to time. Scale bar 100 µm. Whole western blots for 5A and 5B are in Figure S10 .

    Journal: Cancers

    Article Title: Transcriptional Regulation of CCL2 by PARP1 Is a Driver for Invasiveness in Breast Cancer

    doi: 10.3390/cancers12051317

    Figure Lengend Snippet: PARP1 and CCL2 crosstalk regulate invasiveness in breast cancer. ( A ) Left: Recombinant human CCL2 (rhCCL2) can increase PAR levels in MB-231 cells. MB-231 cells were serum-starved overnight and treated with 50 ng/mL rhCCL2 for 1 h. Western blot showing total PAR level upon treatment. Right: Immunofluorescence images showing PAR (green) in cells after rhCCL2 treatment. An enlarged image in Figure S8 . Merged image with DNA (DAPI) shown on top. Scale bar 20 µm. ( B ) PARP1 inhibitor treatment reduces phosphorylated P44/42 (ERK1/2) levels in MB-231cells. Western blot showing Phospho-p44/42 ERK1/2 (Thr202/Tyr204) and total ERK1/2 after overnight PJ34 treatment followed by 45 min of TNFα. ( C ) Left: Heat map showing transcriptome analysis from MB-231 cells for rhCCL2 (1 h treatment as in A ) and PJ34 (10 µM Overnight treatment) treated groups. The KEGG MAP kinase pathway genes are identified. Red color indicates upregulation with green, showing the downregulation of mRNA transcription. Right: Ingenuity pathway analysis from rhCCL2 (Left column) and PJ34 treated (right column). Enlarged images of ( C ) are in Figure S8 . ( D ) Wound healing assay with simultaneous rhCCL2 and PJ34 treatment on MB-231 cells. Near confluent cells were pretreated with PJ34 before rhCCL2 was added at the dose of 50 ng/mL after wound creation. Treatment was continued for 12 h. The graph on the right shows % wound closure with respect to time. Scale bar 100 µm. Whole western blots for 5A and 5B are in Figure S10 .

    Article Snippet: The following are available online at https://www.mdpi.com/2072-6694/12/5/1317/s1 : Figure S1: Overnight treatment with PJ34 does not cause significant apoptosis, Figure S2: PARP1 inhibition or knockdown affects inflammatory and oncogenic pathways, Figure S3: GSEA leading-edge analysis on significant Gene sets from PJ34-PARPi with 656 overlapping genes, Figure S4: Downregulation of CCL2 upon PARP1 inhibitor Treatment in BRCA negative TNBC cells, Figure S5: PARP1 localization across the CCL2 gene locus on chromosome 17, Figure S6: PARP1 inhibition reduces nuclear P65, Figure S7: Expression of PARP1, P65, CCL2 from TCGA breast cancer dataset, Figure S8: Enlarged images of A,C, Figure S9: Whole western blots in , and , Figure S10: Whole western blots in and , Figure S11.Whole western blots in Figure S6, Figure S12: Enlarged view of C Boyden chamber assay, Table S1: Gene lists for the whole transcriptome analysis.

    Techniques: Recombinant, Western Blot, Immunofluorescence, Wound Healing Assay

    siRNA mediated PARP1 knockdown downregulates CCL2 transcription. ( A ) Western blot for Total PARP1. Knockdown efficiency with PARP1 RNAi is confirmed with two different PARP1 siRNAs in MB-231and BT549 cells. ( B ) Western blot showing the total PAR level in the two breast cancer cells with siRNA mediated PARP1 knockdown. GAPDH is shown as a loading control. ( C ) Relative expression of CCL2 mRNA upon siRNA mediated PARP1 knockdown in MB-231 and ( D ) BT549 cells (Right). For ( C ) and ( D ), RNA was harvested after 72 h post-transfection. siRNA knockdown was performed independently twice. Polymerase chain reaction (PCR) was performed twice in triplicates, data presented as ± S.E.M., * p

    Journal: Cancers

    Article Title: Transcriptional Regulation of CCL2 by PARP1 Is a Driver for Invasiveness in Breast Cancer

    doi: 10.3390/cancers12051317

    Figure Lengend Snippet: siRNA mediated PARP1 knockdown downregulates CCL2 transcription. ( A ) Western blot for Total PARP1. Knockdown efficiency with PARP1 RNAi is confirmed with two different PARP1 siRNAs in MB-231and BT549 cells. ( B ) Western blot showing the total PAR level in the two breast cancer cells with siRNA mediated PARP1 knockdown. GAPDH is shown as a loading control. ( C ) Relative expression of CCL2 mRNA upon siRNA mediated PARP1 knockdown in MB-231 and ( D ) BT549 cells (Right). For ( C ) and ( D ), RNA was harvested after 72 h post-transfection. siRNA knockdown was performed independently twice. Polymerase chain reaction (PCR) was performed twice in triplicates, data presented as ± S.E.M., * p

    Article Snippet: The following are available online at https://www.mdpi.com/2072-6694/12/5/1317/s1 : Figure S1: Overnight treatment with PJ34 does not cause significant apoptosis, Figure S2: PARP1 inhibition or knockdown affects inflammatory and oncogenic pathways, Figure S3: GSEA leading-edge analysis on significant Gene sets from PJ34-PARPi with 656 overlapping genes, Figure S4: Downregulation of CCL2 upon PARP1 inhibitor Treatment in BRCA negative TNBC cells, Figure S5: PARP1 localization across the CCL2 gene locus on chromosome 17, Figure S6: PARP1 inhibition reduces nuclear P65, Figure S7: Expression of PARP1, P65, CCL2 from TCGA breast cancer dataset, Figure S8: Enlarged images of A,C, Figure S9: Whole western blots in , and , Figure S10: Whole western blots in and , Figure S11.Whole western blots in Figure S6, Figure S12: Enlarged view of C Boyden chamber assay, Table S1: Gene lists for the whole transcriptome analysis.

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

    PARP1 and NFκB interaction is important for transcriptional control of CCL2 . ( A ) Quantitative real-time PCR for relative expression of CCL2 mRNA after NFκB p65 inhibitor Bay 11-7081 treatment in MB-231 cells. Cells were rhCCL2-treated at the indicated doses for 4 h. * p = 0.001, ** p = 0.0002, one way ANOVA and Tukey’s comparison with untreated ( B ) Left: Western blot showed activation of NFκB pathway probed with an antibody against phosphorylated P65 S536. MB-231 cells were pretreated with PJ34 overnight, and TNFα was added for 45 min. Right: Electrophoretic mobility shift assay (EMSA) with p65 probe. The nuclear lysate was prepared from PJ34 treated or untreated cells, incubated with p65 specific biotinylated DNA probe. Bound and unbound probes were visualized using HRP-conjugated streptavidin chemiluminescence. ( C ) PARP1 and NFκB interact in MB-231cells. Immunoprecipitation was done with the P65 antibody using nuclear lysate. Lysate and IP fractions were western blotted and probed with PARP1 and P65 specific antibodies. ( D ) Chromatin IP with PARP1 and P65 specific antibody. Fixed chromatin was immunoprecipitated with the antibody, as mentioned earlier, after overnight treatment with 10 µM PJ34. We performed PCR on a region flanking an NFκB binding motif at the CCL2 promoter (Dotted arrows showing the approximate location of the primers). Percent of input shows enrichment. Average of two independent experiments shown. t -test * p

    Journal: Cancers

    Article Title: Transcriptional Regulation of CCL2 by PARP1 Is a Driver for Invasiveness in Breast Cancer

    doi: 10.3390/cancers12051317

    Figure Lengend Snippet: PARP1 and NFκB interaction is important for transcriptional control of CCL2 . ( A ) Quantitative real-time PCR for relative expression of CCL2 mRNA after NFκB p65 inhibitor Bay 11-7081 treatment in MB-231 cells. Cells were rhCCL2-treated at the indicated doses for 4 h. * p = 0.001, ** p = 0.0002, one way ANOVA and Tukey’s comparison with untreated ( B ) Left: Western blot showed activation of NFκB pathway probed with an antibody against phosphorylated P65 S536. MB-231 cells were pretreated with PJ34 overnight, and TNFα was added for 45 min. Right: Electrophoretic mobility shift assay (EMSA) with p65 probe. The nuclear lysate was prepared from PJ34 treated or untreated cells, incubated with p65 specific biotinylated DNA probe. Bound and unbound probes were visualized using HRP-conjugated streptavidin chemiluminescence. ( C ) PARP1 and NFκB interact in MB-231cells. Immunoprecipitation was done with the P65 antibody using nuclear lysate. Lysate and IP fractions were western blotted and probed with PARP1 and P65 specific antibodies. ( D ) Chromatin IP with PARP1 and P65 specific antibody. Fixed chromatin was immunoprecipitated with the antibody, as mentioned earlier, after overnight treatment with 10 µM PJ34. We performed PCR on a region flanking an NFκB binding motif at the CCL2 promoter (Dotted arrows showing the approximate location of the primers). Percent of input shows enrichment. Average of two independent experiments shown. t -test * p

    Article Snippet: The following are available online at https://www.mdpi.com/2072-6694/12/5/1317/s1 : Figure S1: Overnight treatment with PJ34 does not cause significant apoptosis, Figure S2: PARP1 inhibition or knockdown affects inflammatory and oncogenic pathways, Figure S3: GSEA leading-edge analysis on significant Gene sets from PJ34-PARPi with 656 overlapping genes, Figure S4: Downregulation of CCL2 upon PARP1 inhibitor Treatment in BRCA negative TNBC cells, Figure S5: PARP1 localization across the CCL2 gene locus on chromosome 17, Figure S6: PARP1 inhibition reduces nuclear P65, Figure S7: Expression of PARP1, P65, CCL2 from TCGA breast cancer dataset, Figure S8: Enlarged images of A,C, Figure S9: Whole western blots in , and , Figure S10: Whole western blots in and , Figure S11.Whole western blots in Figure S6, Figure S12: Enlarged view of C Boyden chamber assay, Table S1: Gene lists for the whole transcriptome analysis.

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Activation Assay, Electrophoretic Mobility Shift Assay, Incubation, Immunoprecipitation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay

    Modulating intracellular PAR levels affects CCL2 transcription. ( A ) PAR and PARP1 levels after overnight 10 µM PJ34 treatment in MB-231cells. Left: Western bolt showing PARP1 and PAR levels. Right: Relative CCL2 transcript levels after overnight PJ34 treatment in MB-231with PARP1 inhibitor PJ34. ( B ) Secreted CCL2 (pg/mL) in cell-conditioned media after PJ34 treatment. Serum-free cell-conditioned media was collected and subjected to CCL2 ELISA. CCL2 protein levels were normalized to 10 6 cells. ( C ) Left: PAR and PARP1 levels after overnight PARG inhibitor (PDD 00017273) treatment in MB-231 cells. Right: Relative expression of CCL2 mRNA after PARG inhibitor treatment. ( D ) Relative expression of CCL2 mRNA in the presence of PARG inhibitor, PARP1 inhibitor and the combination of the two at 5 µM and 10µM doses respectively, Data ± S.E.M. ** p

    Journal: Cancers

    Article Title: Transcriptional Regulation of CCL2 by PARP1 Is a Driver for Invasiveness in Breast Cancer

    doi: 10.3390/cancers12051317

    Figure Lengend Snippet: Modulating intracellular PAR levels affects CCL2 transcription. ( A ) PAR and PARP1 levels after overnight 10 µM PJ34 treatment in MB-231cells. Left: Western bolt showing PARP1 and PAR levels. Right: Relative CCL2 transcript levels after overnight PJ34 treatment in MB-231with PARP1 inhibitor PJ34. ( B ) Secreted CCL2 (pg/mL) in cell-conditioned media after PJ34 treatment. Serum-free cell-conditioned media was collected and subjected to CCL2 ELISA. CCL2 protein levels were normalized to 10 6 cells. ( C ) Left: PAR and PARP1 levels after overnight PARG inhibitor (PDD 00017273) treatment in MB-231 cells. Right: Relative expression of CCL2 mRNA after PARG inhibitor treatment. ( D ) Relative expression of CCL2 mRNA in the presence of PARG inhibitor, PARP1 inhibitor and the combination of the two at 5 µM and 10µM doses respectively, Data ± S.E.M. ** p

    Article Snippet: The following are available online at https://www.mdpi.com/2072-6694/12/5/1317/s1 : Figure S1: Overnight treatment with PJ34 does not cause significant apoptosis, Figure S2: PARP1 inhibition or knockdown affects inflammatory and oncogenic pathways, Figure S3: GSEA leading-edge analysis on significant Gene sets from PJ34-PARPi with 656 overlapping genes, Figure S4: Downregulation of CCL2 upon PARP1 inhibitor Treatment in BRCA negative TNBC cells, Figure S5: PARP1 localization across the CCL2 gene locus on chromosome 17, Figure S6: PARP1 inhibition reduces nuclear P65, Figure S7: Expression of PARP1, P65, CCL2 from TCGA breast cancer dataset, Figure S8: Enlarged images of A,C, Figure S9: Whole western blots in , and , Figure S10: Whole western blots in and , Figure S11.Whole western blots in Figure S6, Figure S12: Enlarged view of C Boyden chamber assay, Table S1: Gene lists for the whole transcriptome analysis.

    Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Expressing

    PARP1 inhibition resulted in reduced cell proliferation and migration in breast cancer cells. ( A ) Western blots for total levels of PARP1 and PolyADP Ribose (PAR) in a panel of breast cancer cells. Triple-negative cell lines are on the left. GAPDH is used as a loading control. Whole western blots for 1A are in Figure S9 ( B ) Left: Cell proliferation upon PJ34 PARP inhibitor treatment in MB-231 cells at 48 h and 72 h. Right: Colony formation assay with PJ34 treated MB-231 cells in low attachment plates for 7 days. Scale bar 10 µm ( C ) Left: Wound healing assay in the presence of PJ34 in MB-231 cells. Right: Boyden chamber Invasion assay in the presence of PJ34 inhibitor using MB-231 cells. Cells were incubated with PJ34 for the duration of the experiment. Experiments were done in duplicate two times. Dark objects on the pictures are cells. One arrow is shown in each panel, pointing to one of the cells. * p

    Journal: Cancers

    Article Title: Transcriptional Regulation of CCL2 by PARP1 Is a Driver for Invasiveness in Breast Cancer

    doi: 10.3390/cancers12051317

    Figure Lengend Snippet: PARP1 inhibition resulted in reduced cell proliferation and migration in breast cancer cells. ( A ) Western blots for total levels of PARP1 and PolyADP Ribose (PAR) in a panel of breast cancer cells. Triple-negative cell lines are on the left. GAPDH is used as a loading control. Whole western blots for 1A are in Figure S9 ( B ) Left: Cell proliferation upon PJ34 PARP inhibitor treatment in MB-231 cells at 48 h and 72 h. Right: Colony formation assay with PJ34 treated MB-231 cells in low attachment plates for 7 days. Scale bar 10 µm ( C ) Left: Wound healing assay in the presence of PJ34 in MB-231 cells. Right: Boyden chamber Invasion assay in the presence of PJ34 inhibitor using MB-231 cells. Cells were incubated with PJ34 for the duration of the experiment. Experiments were done in duplicate two times. Dark objects on the pictures are cells. One arrow is shown in each panel, pointing to one of the cells. * p

    Article Snippet: The following are available online at https://www.mdpi.com/2072-6694/12/5/1317/s1 : Figure S1: Overnight treatment with PJ34 does not cause significant apoptosis, Figure S2: PARP1 inhibition or knockdown affects inflammatory and oncogenic pathways, Figure S3: GSEA leading-edge analysis on significant Gene sets from PJ34-PARPi with 656 overlapping genes, Figure S4: Downregulation of CCL2 upon PARP1 inhibitor Treatment in BRCA negative TNBC cells, Figure S5: PARP1 localization across the CCL2 gene locus on chromosome 17, Figure S6: PARP1 inhibition reduces nuclear P65, Figure S7: Expression of PARP1, P65, CCL2 from TCGA breast cancer dataset, Figure S8: Enlarged images of A,C, Figure S9: Whole western blots in , and , Figure S10: Whole western blots in and , Figure S11.Whole western blots in Figure S6, Figure S12: Enlarged view of C Boyden chamber assay, Table S1: Gene lists for the whole transcriptome analysis.

    Techniques: Inhibition, Migration, Western Blot, Colony Assay, Wound Healing Assay, Invasion Assay, Incubation

    LSS-11 enhances cell apoptosis in A549/T cells. ( a ) Percentage of apoptotic A549/T cells were analyzed by flow cytometry after exposure to 0.5 μM LSS-11 at indicated times. Protein levels of DR5, PARP-1, cleaved PARP1 ( b ), Bax, and Bcl2 ( c ) after LSS-11 treatment for 24 h. Quantification of protein bands was shown as bar graph in the right panels. * p

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: The Novel Triazolonaphthalimide Derivative LSS-11 Synergizes the Anti-Proliferative Effect of Paclitaxel via STAT3-Dependent MDR1 and MRP1 Downregulation in Chemoresistant Lung Cancer Cells

    doi: 10.3390/molecules22111822

    Figure Lengend Snippet: LSS-11 enhances cell apoptosis in A549/T cells. ( a ) Percentage of apoptotic A549/T cells were analyzed by flow cytometry after exposure to 0.5 μM LSS-11 at indicated times. Protein levels of DR5, PARP-1, cleaved PARP1 ( b ), Bax, and Bcl2 ( c ) after LSS-11 treatment for 24 h. Quantification of protein bands was shown as bar graph in the right panels. * p

    Article Snippet: Primary antibodies against DR5, PARP1, cleaved PARP1, Bax, Bcl2, GAPDH, and secondary antibodies including FITC-linked anti-rabbit and HRP-linked anti-rabbit or anti-mouse were obtained from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Flow Cytometry, Cytometry

    Decreased levels of DNA repair proteins and measurement of oxidative DNA damage and PARP1 activity in neuronal nuclei after OGD. (a) The levels of DNA repair proteins PARP1, XRCC1, OGG1, and APE1 in nuclear extracts of neurons exposed to control (lane

    Journal: Journal of neurochemistry

    Article Title: INTRANUCLEAR MATRIX METALLOPROTEINASES PROMOTE DNA DAMAGE AND APOPTOSIS INDUCED BY OXYGEN–GLUCOSE DEPRIVATION IN NEURONS

    doi: 10.1111/j.1471-4159.2009.06433.x

    Figure Lengend Snippet: Decreased levels of DNA repair proteins and measurement of oxidative DNA damage and PARP1 activity in neuronal nuclei after OGD. (a) The levels of DNA repair proteins PARP1, XRCC1, OGG1, and APE1 in nuclear extracts of neurons exposed to control (lane

    Article Snippet: Separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore) and specific proteins were detected using antibodies against PARP1 (Cell Signaling Technology, Inc., Danvers, MA, USA), XRCC1 (Sigma–Aldrich), 8-oxoguanine DNA glycosylase 1 (OGG1) (Novus Biologicals, Littleton, CO, USA), and apurinic/apyrimidinic (AP) DNA endonuclease 1 (APE1) (Novus Biologicals).

    Techniques: Activity Assay

    Effects of siRNAs specific to CXXC5 on cellular growth. MCF7 cells grown in 10% CD-FBS containing medium for 48 h were transiently transfected without (UT) or with 10 nM CtS or siRNA#10 in the absence (0.01% ethanol) or the presence of 10 −8 M E2 up to 72 h. ( a ) Cells were subjected to cell counting using a hemocytometer. Results, as the mean ± S.E. of three independent determinations, depict fold change in cellular growth compared with those observed with cells at time 0 in the absence of E2, which is set to 1. In ( a–f ) the asterisk with a superscript “a” indicates significant difference from the corresponding ethanol-treated group (-E2); whereas superscript “b” depicts a significant difference from CtS transfected cells in the presence of E2. ( b ) The cDNA library generated from total RNA (50 ng) isolated from transfected cells for 48 h was subjected to qPCR using a primer set specific to CXXC5. ( c ) Nuclear extracts from untransfected (UT) or transfected cells were subjected to WB using an antibody for CXXC5, ERα or HDAC1. NS denotes non-specific protein; Molecular mass in KDa is indicated. Transfected cells were subjected to cell counting ( d ) and MTT assay ( e ). In ( b , d , e) , results depicting the mean ± S.E. of three independent determinations indicate fold change in mRNA levels ( b ) or cell numbers ( d , e ) compared with CtS in the absence of E2, which is set to 1. Transfected MCF7 cells were subjected to flow cytometry ( f ) with results depicting the percent of cells in the G1, G2 and S phases, or to Annexin V assay followed by flow cytometry ( g ). In ( f , g ), results are the mean ± SEM of three independent experiments. ( h i ) Untransfected (UT) or transfected cells were also subjected to the cell death inducer camptothecin (2 nM) for 24 h without (−E2) or with E2 (E2). Cells were then subjected to Annexin V assay ( h ) with results as the mean ± S.E. of three independent experiments depicting percent change in apoptotic (upper right quadrant) and death (upper left quadrant) cells, or to WB ( i ) using a PARP1-specific antibody. P and CP denote the uncut and cut PARP1, respectively.

    Journal: Scientific Reports

    Article Title: CXXC5 as an unmethylated CpG dinucleotide binding protein contributes to estrogen-mediated cellular proliferation

    doi: 10.1038/s41598-020-62912-0

    Figure Lengend Snippet: Effects of siRNAs specific to CXXC5 on cellular growth. MCF7 cells grown in 10% CD-FBS containing medium for 48 h were transiently transfected without (UT) or with 10 nM CtS or siRNA#10 in the absence (0.01% ethanol) or the presence of 10 −8 M E2 up to 72 h. ( a ) Cells were subjected to cell counting using a hemocytometer. Results, as the mean ± S.E. of three independent determinations, depict fold change in cellular growth compared with those observed with cells at time 0 in the absence of E2, which is set to 1. In ( a–f ) the asterisk with a superscript “a” indicates significant difference from the corresponding ethanol-treated group (-E2); whereas superscript “b” depicts a significant difference from CtS transfected cells in the presence of E2. ( b ) The cDNA library generated from total RNA (50 ng) isolated from transfected cells for 48 h was subjected to qPCR using a primer set specific to CXXC5. ( c ) Nuclear extracts from untransfected (UT) or transfected cells were subjected to WB using an antibody for CXXC5, ERα or HDAC1. NS denotes non-specific protein; Molecular mass in KDa is indicated. Transfected cells were subjected to cell counting ( d ) and MTT assay ( e ). In ( b , d , e) , results depicting the mean ± S.E. of three independent determinations indicate fold change in mRNA levels ( b ) or cell numbers ( d , e ) compared with CtS in the absence of E2, which is set to 1. Transfected MCF7 cells were subjected to flow cytometry ( f ) with results depicting the percent of cells in the G1, G2 and S phases, or to Annexin V assay followed by flow cytometry ( g ). In ( f , g ), results are the mean ± SEM of three independent experiments. ( h i ) Untransfected (UT) or transfected cells were also subjected to the cell death inducer camptothecin (2 nM) for 24 h without (−E2) or with E2 (E2). Cells were then subjected to Annexin V assay ( h ) with results as the mean ± S.E. of three independent experiments depicting percent change in apoptotic (upper right quadrant) and death (upper left quadrant) cells, or to WB ( i ) using a PARP1-specific antibody. P and CP denote the uncut and cut PARP1, respectively.

    Article Snippet: Using the experimental approach described here for Annexin V assay, we also carried out WB for the cleavage of Poly(ADP-ribose) Polymerase (PARP) as an indication of apoptosis, with a PARP1-specific antibody (9542, Cell Signaling Tech.) that also detects the cleaved PARP.

    Techniques: Transfection, Cell Counting, cDNA Library Assay, Generated, Isolation, Real-time Polymerase Chain Reaction, Western Blot, MTT Assay, Flow Cytometry, Annexin V Assay

    Time-dependent activation of PARP upon H 2 O 2 -treatment in U937 cells is accelerated in the presence of isoproterenol. (A) The earliest detectable level of PARylation occurring at 4 minutes postchallenge with H 2 O 2 (400 μ M). (B) Pretreatment of U937

    Journal: Molecular Pharmacology

    Article Title: Regulation of Mitochondrial Poly(ADP-Ribose) Polymerase Activation by the β-Adrenoceptor/cAMP/Protein Kinase A Axis during Oxidative Stress

    doi: 10.1124/mol.114.094318

    Figure Lengend Snippet: Time-dependent activation of PARP upon H 2 O 2 -treatment in U937 cells is accelerated in the presence of isoproterenol. (A) The earliest detectable level of PARylation occurring at 4 minutes postchallenge with H 2 O 2 (400 μ M). (B) Pretreatment of U937

    Article Snippet: Western blotting analysis was carried out as previously described ( ) using anti-PARP1 antibody (Cell Signaling Technology, Beverley, MA), anti- β -receptor antibody (Abcam, Cambridge, MA), anti-protein kinase A (R & D Systems, Minneapolis, MN), anti- β -actin–horseradish peroxidase (HRP) conjugate (Santa Cruz Biotechnology, Inc., Dallas, TX), anti-rabbit-IgG (Cell Signaling Technology), and anti-mouse-IgG (Cell Signaling Technology).

    Techniques: Activation Assay

    The β -adrenoceptor/cAMP/PKA system regulates PARP activity in C2C12 cell line. The β -adrenergic receptor antagonist propranolol (PP) (A) or the protein kinase inhibitor PKAi (B) decrease cellular PARylation induced by H 2 O 2 in C2C12 cells.

    Journal: Molecular Pharmacology

    Article Title: Regulation of Mitochondrial Poly(ADP-Ribose) Polymerase Activation by the β-Adrenoceptor/cAMP/Protein Kinase A Axis during Oxidative Stress

    doi: 10.1124/mol.114.094318

    Figure Lengend Snippet: The β -adrenoceptor/cAMP/PKA system regulates PARP activity in C2C12 cell line. The β -adrenergic receptor antagonist propranolol (PP) (A) or the protein kinase inhibitor PKAi (B) decrease cellular PARylation induced by H 2 O 2 in C2C12 cells.

    Article Snippet: Western blotting analysis was carried out as previously described ( ) using anti-PARP1 antibody (Cell Signaling Technology, Beverley, MA), anti- β -receptor antibody (Abcam, Cambridge, MA), anti-protein kinase A (R & D Systems, Minneapolis, MN), anti- β -actin–horseradish peroxidase (HRP) conjugate (Santa Cruz Biotechnology, Inc., Dallas, TX), anti-rabbit-IgG (Cell Signaling Technology), and anti-mouse-IgG (Cell Signaling Technology).

    Techniques: Activity Assay

    PARP1 is specifically phosphorylated by PKA on serine residues in U937 cells during oxidative stress. (A) Serine-specific phosphorylation of PARP1, as evidenced by in situ PLA analysis. Control experiments show the auto-PARylation of PARP1. (B) H 2 O 2 induces

    Journal: Molecular Pharmacology

    Article Title: Regulation of Mitochondrial Poly(ADP-Ribose) Polymerase Activation by the β-Adrenoceptor/cAMP/Protein Kinase A Axis during Oxidative Stress

    doi: 10.1124/mol.114.094318

    Figure Lengend Snippet: PARP1 is specifically phosphorylated by PKA on serine residues in U937 cells during oxidative stress. (A) Serine-specific phosphorylation of PARP1, as evidenced by in situ PLA analysis. Control experiments show the auto-PARylation of PARP1. (B) H 2 O 2 induces

    Article Snippet: Western blotting analysis was carried out as previously described ( ) using anti-PARP1 antibody (Cell Signaling Technology, Beverley, MA), anti- β -receptor antibody (Abcam, Cambridge, MA), anti-protein kinase A (R & D Systems, Minneapolis, MN), anti- β -actin–horseradish peroxidase (HRP) conjugate (Santa Cruz Biotechnology, Inc., Dallas, TX), anti-rabbit-IgG (Cell Signaling Technology), and anti-mouse-IgG (Cell Signaling Technology).

    Techniques: In Situ, Proximity Ligation Assay

    Phosphorylation of PARP1 requires its auto-PARylation. (A) In vitro phosphorylation assay with PKA and PARP1 recombinants proteins was analyzed by Western blotting. The catalytic activity of PKA increased by the addition of cAMP or NAD + /DNA and decreased

    Journal: Molecular Pharmacology

    Article Title: Regulation of Mitochondrial Poly(ADP-Ribose) Polymerase Activation by the β-Adrenoceptor/cAMP/Protein Kinase A Axis during Oxidative Stress

    doi: 10.1124/mol.114.094318

    Figure Lengend Snippet: Phosphorylation of PARP1 requires its auto-PARylation. (A) In vitro phosphorylation assay with PKA and PARP1 recombinants proteins was analyzed by Western blotting. The catalytic activity of PKA increased by the addition of cAMP or NAD + /DNA and decreased

    Article Snippet: Western blotting analysis was carried out as previously described ( ) using anti-PARP1 antibody (Cell Signaling Technology, Beverley, MA), anti- β -receptor antibody (Abcam, Cambridge, MA), anti-protein kinase A (R & D Systems, Minneapolis, MN), anti- β -actin–horseradish peroxidase (HRP) conjugate (Santa Cruz Biotechnology, Inc., Dallas, TX), anti-rabbit-IgG (Cell Signaling Technology), and anti-mouse-IgG (Cell Signaling Technology).

    Techniques: In Vitro, Phosphorylation Assay, Western Blot, Activity Assay

    Time course of PARP1 activation in the extranuclear and nuclear compartments of U937 cells subjected to oxidative stress. (A) H 2 O 2 -induced PARP activation appears initially extranuclearly (10–60 minutes), while at subsequent time points nuclear

    Journal: Molecular Pharmacology

    Article Title: Regulation of Mitochondrial Poly(ADP-Ribose) Polymerase Activation by the β-Adrenoceptor/cAMP/Protein Kinase A Axis during Oxidative Stress

    doi: 10.1124/mol.114.094318

    Figure Lengend Snippet: Time course of PARP1 activation in the extranuclear and nuclear compartments of U937 cells subjected to oxidative stress. (A) H 2 O 2 -induced PARP activation appears initially extranuclearly (10–60 minutes), while at subsequent time points nuclear

    Article Snippet: Western blotting analysis was carried out as previously described ( ) using anti-PARP1 antibody (Cell Signaling Technology, Beverley, MA), anti- β -receptor antibody (Abcam, Cambridge, MA), anti-protein kinase A (R & D Systems, Minneapolis, MN), anti- β -actin–horseradish peroxidase (HRP) conjugate (Santa Cruz Biotechnology, Inc., Dallas, TX), anti-rabbit-IgG (Cell Signaling Technology), and anti-mouse-IgG (Cell Signaling Technology).

    Techniques: Activation Assay

    Proposed model of PKA-PARP1 interactions in the early stage of oxidative stress in U937 cells. Upon oxidative stress, mitochondrial DNA strand breakage occurs, which activates PARP1 in the mitochondria. (A) The cAMP/PKA axis is stimulated by β

    Journal: Molecular Pharmacology

    Article Title: Regulation of Mitochondrial Poly(ADP-Ribose) Polymerase Activation by the β-Adrenoceptor/cAMP/Protein Kinase A Axis during Oxidative Stress

    doi: 10.1124/mol.114.094318

    Figure Lengend Snippet: Proposed model of PKA-PARP1 interactions in the early stage of oxidative stress in U937 cells. Upon oxidative stress, mitochondrial DNA strand breakage occurs, which activates PARP1 in the mitochondria. (A) The cAMP/PKA axis is stimulated by β

    Article Snippet: Western blotting analysis was carried out as previously described ( ) using anti-PARP1 antibody (Cell Signaling Technology, Beverley, MA), anti- β -receptor antibody (Abcam, Cambridge, MA), anti-protein kinase A (R & D Systems, Minneapolis, MN), anti- β -actin–horseradish peroxidase (HRP) conjugate (Santa Cruz Biotechnology, Inc., Dallas, TX), anti-rabbit-IgG (Cell Signaling Technology), and anti-mouse-IgG (Cell Signaling Technology).

    Techniques:

    β -adrenoceptor/cAMP/PKA signaling regulates PARP activation in U937 cells during oxidative stress. The β -adrenoceptor antagonists propranolol (PP) (A), the adenylyl cyclase inhibitor DDA (B), or the PKA inhibitor PKAi (C) decrease cellular

    Journal: Molecular Pharmacology

    Article Title: Regulation of Mitochondrial Poly(ADP-Ribose) Polymerase Activation by the β-Adrenoceptor/cAMP/Protein Kinase A Axis during Oxidative Stress

    doi: 10.1124/mol.114.094318

    Figure Lengend Snippet: β -adrenoceptor/cAMP/PKA signaling regulates PARP activation in U937 cells during oxidative stress. The β -adrenoceptor antagonists propranolol (PP) (A), the adenylyl cyclase inhibitor DDA (B), or the PKA inhibitor PKAi (C) decrease cellular

    Article Snippet: Western blotting analysis was carried out as previously described ( ) using anti-PARP1 antibody (Cell Signaling Technology, Beverley, MA), anti- β -receptor antibody (Abcam, Cambridge, MA), anti-protein kinase A (R & D Systems, Minneapolis, MN), anti- β -actin–horseradish peroxidase (HRP) conjugate (Santa Cruz Biotechnology, Inc., Dallas, TX), anti-rabbit-IgG (Cell Signaling Technology), and anti-mouse-IgG (Cell Signaling Technology).

    Techniques: Activation Assay

    Figure 8. Schematic representation of the protective effects of rapamycin against AMA toxicity. AMA induces PARP1 cleavage and CASP3 activation, decreases respiration, mitochondrial membrane potential as well as mediates the accumulation of ubiquitinated

    Journal: Autophagy

    Article Title: Upregulated autophagy protects cardiomyocytes from oxidative stress-induced toxicity

    doi: 10.4161/auto.22971

    Figure Lengend Snippet: Figure 8. Schematic representation of the protective effects of rapamycin against AMA toxicity. AMA induces PARP1 cleavage and CASP3 activation, decreases respiration, mitochondrial membrane potential as well as mediates the accumulation of ubiquitinated

    Article Snippet: The primary antibodies used are as follows: rabbit anti-LC3B (Cell Signaling, 2775), rabbit anti-BECN1 (Cell Signaling, 3738), rabbit anti-PARP1 (Cell Signaling, 9542), rabbit anti-CASP3 (Cell Signaling, 9662), rabbit anti-phospho-RPS6 (Cell Signaling, 2211), mouse anti-RPS6 (Cell Signaling, 2317), rabbit anti-ubiquitin (Cell Signaling, 3936), rabbit anti-SQSTM1 (Cell Signaling, 5114), rabbit anti-cyt c (Cell Signaling, 4272), mouse anti-ATG5 (Sigma, A2859), mouse anti-VDAC1 (Mitoscences, MSA03), rabbit anti-TUBB/β-tubulin (Sigma, T2200) and GAPDH-HRP (Sigma, G9295).

    Techniques: Activation Assay

    Western blot analysis for caspases 9 and 3, cleaved caspases 9 and 8 (cCaspase), PARP1 and cleaved PARP1 (cPARP1) in U87 ( a ), IC ( b ) and LN229 ( c ) cells after 48 h and 72 h exposure of JS-K (1–5 μ M). 25 μ g of protein lysates were separated with SDS-PAGE and probed with the different antibodies. Caspase 9 and 8 are expressed and cleaved equally in all three cell lines and both in untreated and DMSO controls. No cleavage and no decrease in protein level of caspase 3 can be shown. The figures shown are representative for three independent experiments

    Journal: Cell Death & Disease

    Article Title: Nitric oxide released from JS-K induces cell death by mitotic catastrophe as part of necrosis in glioblastoma multiforme

    doi: 10.1038/cddis.2016.254

    Figure Lengend Snippet: Western blot analysis for caspases 9 and 3, cleaved caspases 9 and 8 (cCaspase), PARP1 and cleaved PARP1 (cPARP1) in U87 ( a ), IC ( b ) and LN229 ( c ) cells after 48 h and 72 h exposure of JS-K (1–5 μ M). 25 μ g of protein lysates were separated with SDS-PAGE and probed with the different antibodies. Caspase 9 and 8 are expressed and cleaved equally in all three cell lines and both in untreated and DMSO controls. No cleavage and no decrease in protein level of caspase 3 can be shown. The figures shown are representative for three independent experiments

    Article Snippet: Blots were incubated with primary antibodies anti-PARP1 (1:1000 Cell Signaling Technology, Inc., Danvers, MA, USA), anti-caspase 3, 8, 9 (1:500 Cell Signaling Technology, Inc.), anti-GAPDH (1:10000 abcam, Cambridge, UK) overnight at 4 °C.

    Techniques: Western Blot, SDS Page

    Deletion of PARP1 fully inhibits H 2 O 2 production, mitochondrial depolarization, caspase activation, and loss of ATP cell content in APO866-treated Jurkat cells. Detection of cytosolic ( A ), mitochondrial ( B ) superoxide anions, NO ( C ), H 2 O 2 generation ( D ) and MMP ( E ), and ATP cell content ( G ) in APO866-treated WT or KO Jurkat cells were monitored as described in Figure 1. ( F ) Caspase 3 activation was assessed in WT (or PARP1KO) Jurkat cells treated with 10 nM APO866 for 96 h using a fluorescent specific probe for activated forms of CAS P3 and flow cytometry.

    Journal: Oncotarget

    Article Title: Reactive oxygen/nitrogen species contribute substantially to the antileukemia effect of APO866, a NAD lowering agent

    doi: 10.18632/oncotarget.27336

    Figure Lengend Snippet: Deletion of PARP1 fully inhibits H 2 O 2 production, mitochondrial depolarization, caspase activation, and loss of ATP cell content in APO866-treated Jurkat cells. Detection of cytosolic ( A ), mitochondrial ( B ) superoxide anions, NO ( C ), H 2 O 2 generation ( D ) and MMP ( E ), and ATP cell content ( G ) in APO866-treated WT or KO Jurkat cells were monitored as described in Figure 1. ( F ) Caspase 3 activation was assessed in WT (or PARP1KO) Jurkat cells treated with 10 nM APO866 for 96 h using a fluorescent specific probe for activated forms of CAS P3 and flow cytometry.

    Article Snippet: The mouse anti-PARP1 and the rabbit anti-actin antibodies were from Cell Signaling (9546 and 4970 respectively).

    Techniques: Activation Assay, Flow Cytometry, Cytometry

    The integrity of PARP1 status is required for the anti-leukemia activity of APO866. Cell lines and primary cells from different hematological malignancies were treated without or with 10 nM of APO866 in the presence or absence of a pharmacological inhibition ( A – B ) or genetic deletion ( D ) of PARP1 and cell death assessed as described in Figure 2. PARP1 was knocked out (KO) in wild-type (WT) Jurkat cells using CRISPR/Cas9 technology. Loss of expression was confirmed by Western blotting ( C ).

    Journal: Oncotarget

    Article Title: Reactive oxygen/nitrogen species contribute substantially to the antileukemia effect of APO866, a NAD lowering agent

    doi: 10.18632/oncotarget.27336

    Figure Lengend Snippet: The integrity of PARP1 status is required for the anti-leukemia activity of APO866. Cell lines and primary cells from different hematological malignancies were treated without or with 10 nM of APO866 in the presence or absence of a pharmacological inhibition ( A – B ) or genetic deletion ( D ) of PARP1 and cell death assessed as described in Figure 2. PARP1 was knocked out (KO) in wild-type (WT) Jurkat cells using CRISPR/Cas9 technology. Loss of expression was confirmed by Western blotting ( C ).

    Article Snippet: The mouse anti-PARP1 and the rabbit anti-actin antibodies were from Cell Signaling (9546 and 4970 respectively).

    Techniques: Activity Assay, Inhibition, CRISPR, Expressing, Western Blot

    Exogenous supplementation of H 2 O 2 or etoposide potentiates the anti-leukemia activity of APO866. APO866-treated WT or PARP1 KO cells from different human leukemia exposed to H 2 O 2 ( A ) or not ( B ), or etoposide ( C and D ) alone or in combination for 24 hours (H 2 O 2 ) or 96 hours (Etoposide). Cell death was assessed as described in Figure 2. Data are mean ± SD, n = 3; ** p

    Journal: Oncotarget

    Article Title: Reactive oxygen/nitrogen species contribute substantially to the antileukemia effect of APO866, a NAD lowering agent

    doi: 10.18632/oncotarget.27336

    Figure Lengend Snippet: Exogenous supplementation of H 2 O 2 or etoposide potentiates the anti-leukemia activity of APO866. APO866-treated WT or PARP1 KO cells from different human leukemia exposed to H 2 O 2 ( A ) or not ( B ), or etoposide ( C and D ) alone or in combination for 24 hours (H 2 O 2 ) or 96 hours (Etoposide). Cell death was assessed as described in Figure 2. Data are mean ± SD, n = 3; ** p

    Article Snippet: The mouse anti-PARP1 and the rabbit anti-actin antibodies were from Cell Signaling (9546 and 4970 respectively).

    Techniques: Activity Assay

    Modulation of NKG2DLs. a , b , Expression of NKG2DLs after treatment of bulk AML cells with retinoic acid (ATRA, 1 μM), valproic acid (VPA, 2 μM), 5-azacytidine (5 μM), AG-14361 (20 μM) or veliparib (10 μM). Quantified summarized fold-changes in CD34 + NKG2DL + and CD34 − NKG2DL + populations after 24 h of in vitro treatment ( a , n = 3 cases of AML; b , n = 11 cases of AML). All analysed in technical triplicates; DMSO 0.2% or PBS were used as carrier controls. c , Release of soluble NKG2DLs from sorted CD34 + and corresponding CD34 − AML subpopulations (‘shedding assays’). n = 8 cases of AML; mean results with s.d. are shown. d – g , Baseline mRNA expression of different individual NKG2DLs and their variants. d , Relative expression of single ligands; e , relative summarized ligand expression. n = 10 patients with AML. f , g , Induction of NKG2DL mRNAs after PARP1 inhibition using PARP1 siRNAs ( f , 24 h in vitro treatment; control, scrambled non-coding siRNAs) or AG-14361 ( g , 20 μM, 24 h in vitro treatment; control, DMSO carrier (0.2%)). Fold changes in relative expression levels of mRNA of individual ligands compared to control treatments in individual patients are shown, as indicated. Note that heterogeneous NKG2DLs are upregulated upon PARP inhibition in different cases of AML. Statistical analyses were performed using a two-sided Mann–Whitney U test ( c ) or a two-sided Student’s t -test ( a , b , d – g ). Centre values represent mean, error bars represent s.d.

    Journal: Nature

    Article Title: Absence of NKG2D ligands defines leukaemia stem cells and mediates their immune evasion

    doi: 10.1038/s41586-019-1410-1

    Figure Lengend Snippet: Modulation of NKG2DLs. a , b , Expression of NKG2DLs after treatment of bulk AML cells with retinoic acid (ATRA, 1 μM), valproic acid (VPA, 2 μM), 5-azacytidine (5 μM), AG-14361 (20 μM) or veliparib (10 μM). Quantified summarized fold-changes in CD34 + NKG2DL + and CD34 − NKG2DL + populations after 24 h of in vitro treatment ( a , n = 3 cases of AML; b , n = 11 cases of AML). All analysed in technical triplicates; DMSO 0.2% or PBS were used as carrier controls. c , Release of soluble NKG2DLs from sorted CD34 + and corresponding CD34 − AML subpopulations (‘shedding assays’). n = 8 cases of AML; mean results with s.d. are shown. d – g , Baseline mRNA expression of different individual NKG2DLs and their variants. d , Relative expression of single ligands; e , relative summarized ligand expression. n = 10 patients with AML. f , g , Induction of NKG2DL mRNAs after PARP1 inhibition using PARP1 siRNAs ( f , 24 h in vitro treatment; control, scrambled non-coding siRNAs) or AG-14361 ( g , 20 μM, 24 h in vitro treatment; control, DMSO carrier (0.2%)). Fold changes in relative expression levels of mRNA of individual ligands compared to control treatments in individual patients are shown, as indicated. Note that heterogeneous NKG2DLs are upregulated upon PARP inhibition in different cases of AML. Statistical analyses were performed using a two-sided Mann–Whitney U test ( c ) or a two-sided Student’s t -test ( a , b , d – g ). Centre values represent mean, error bars represent s.d.

    Article Snippet: Immunoblots were performed according to standard procedures using anti-human PARP1 (#9542S) and anti β-actin (#3700S) or anti GAPDH (#5174P) antibodies and HRP-linked secondary reagents (#7074S, anti-rabbit IgG and #7076S, anti-mouse IgG, all from Cell Signaling Technology), and quantified by Fiji software.

    Techniques: Expressing, In Vitro, Inhibition, MANN-WHITNEY

    PARP1 in human AML: expression, association with clinical outcome and induction of NKG2DLs after PARP1 inhibition. a , Survival analysis of patients with AML with high (above median, red line) versus low (below median, blue line) expression of PARP1 mRNA (TCGA, n = 179 patients). b , c , Immunoblot analyses of expression of PARP1 protein in NKG2DL − compared to NKG2DL + AML cells ( b ) and CD34 + compared to CD34 − AML cells ( c ), sorted from the same patient samples. n = 9 for b , n = 3 for c . d – h , Treatment with individual ( d , n = 3; e , n = 12; f , no. 42) or pooled ( g , h , n = 3) PARP1 siRNAs for 24 h inhibits expression of the PARP1 gene ( d , g ; qRT–PCR, GAPDH used as housekeeping control) and induces surface expression of NKG2DLs on human CD34 + AML cells ( e , f , h , no. 38) (control, scrambled non-coding PARP1 siRNAs). Each dot represents a different patient analysed in technical triplicates. i , Analysis of expression of NKG2DLs on engrafted human AML subpopulations (percentage of NKG2DL + within CD33 + , CD33 + CD34 + and CD33 + CD34 − subpopulations derived from mouse bone marrow) after five days of in vivo treatment with AG-14361 or DMSO ( n = 10 mice per group, n = 3 cases of AML). j , k , Analysis of surface expression of NKG2DLs after in vitro treatment with AG-14361 (20 μM, 24 h) or DMSO in sorted CD34 + and corresponding CD34 − AML cells ( j , left, representative results; right, summarized fold changes of NKG2DL + cells in AG-14361 versus DMSO cultured cells; n = 3 cases of AML) or in bulk AML cells from non-CD34-expressing cases of AML ( k , n = 10 cases of AML). Summarized fold changes in NKG2DL + AML cells in PARP inhibition versus corresponding control conditions are shown. l , m , Sorted NKG2DL − (top panels) and corresponding NKG2DL + (bottom panels) AML subpopulations were treated for 24 h with AG-14361 (20 μM) or DMSO (0.2%) and analysed by flow cytometry for MICA, MICB, ULBP1 or ULBP2, ULBP5 or ULBP6 surface expression ( l , SFI; no 16, 35 and 151) and using DAPI ( m ) to determine viability (left) and absolute cell numbers (right) ( n = 5 cases of AML). Mean values of technical triplicate analyses are shown. n , Corresponding analyses with healthy CD34 + cells from n = 3 samples of cord blood. o , Leukaemic bone marrow infiltration after in vivo treatment with AG-14361 (days 1 to 5, 10–15 mg kg −1 day −1 ) or DMSO (days 1 to 5, 20%) in n = 10 mice for each group. Statistical analyses were performed using a log-rank (Mantel–Cox) test ( a ), a two-sided Mann–Whitney U test ( k , o ) or a two-sided Student’s t -test (all other plots). Each dot represents the mean of technical triplicates per patient sample. Centre values represent mean, error bars represent s.d.

    Journal: Nature

    Article Title: Absence of NKG2D ligands defines leukaemia stem cells and mediates their immune evasion

    doi: 10.1038/s41586-019-1410-1

    Figure Lengend Snippet: PARP1 in human AML: expression, association with clinical outcome and induction of NKG2DLs after PARP1 inhibition. a , Survival analysis of patients with AML with high (above median, red line) versus low (below median, blue line) expression of PARP1 mRNA (TCGA, n = 179 patients). b , c , Immunoblot analyses of expression of PARP1 protein in NKG2DL − compared to NKG2DL + AML cells ( b ) and CD34 + compared to CD34 − AML cells ( c ), sorted from the same patient samples. n = 9 for b , n = 3 for c . d – h , Treatment with individual ( d , n = 3; e , n = 12; f , no. 42) or pooled ( g , h , n = 3) PARP1 siRNAs for 24 h inhibits expression of the PARP1 gene ( d , g ; qRT–PCR, GAPDH used as housekeeping control) and induces surface expression of NKG2DLs on human CD34 + AML cells ( e , f , h , no. 38) (control, scrambled non-coding PARP1 siRNAs). Each dot represents a different patient analysed in technical triplicates. i , Analysis of expression of NKG2DLs on engrafted human AML subpopulations (percentage of NKG2DL + within CD33 + , CD33 + CD34 + and CD33 + CD34 − subpopulations derived from mouse bone marrow) after five days of in vivo treatment with AG-14361 or DMSO ( n = 10 mice per group, n = 3 cases of AML). j , k , Analysis of surface expression of NKG2DLs after in vitro treatment with AG-14361 (20 μM, 24 h) or DMSO in sorted CD34 + and corresponding CD34 − AML cells ( j , left, representative results; right, summarized fold changes of NKG2DL + cells in AG-14361 versus DMSO cultured cells; n = 3 cases of AML) or in bulk AML cells from non-CD34-expressing cases of AML ( k , n = 10 cases of AML). Summarized fold changes in NKG2DL + AML cells in PARP inhibition versus corresponding control conditions are shown. l , m , Sorted NKG2DL − (top panels) and corresponding NKG2DL + (bottom panels) AML subpopulations were treated for 24 h with AG-14361 (20 μM) or DMSO (0.2%) and analysed by flow cytometry for MICA, MICB, ULBP1 or ULBP2, ULBP5 or ULBP6 surface expression ( l , SFI; no 16, 35 and 151) and using DAPI ( m ) to determine viability (left) and absolute cell numbers (right) ( n = 5 cases of AML). Mean values of technical triplicate analyses are shown. n , Corresponding analyses with healthy CD34 + cells from n = 3 samples of cord blood. o , Leukaemic bone marrow infiltration after in vivo treatment with AG-14361 (days 1 to 5, 10–15 mg kg −1 day −1 ) or DMSO (days 1 to 5, 20%) in n = 10 mice for each group. Statistical analyses were performed using a log-rank (Mantel–Cox) test ( a ), a two-sided Mann–Whitney U test ( k , o ) or a two-sided Student’s t -test (all other plots). Each dot represents the mean of technical triplicates per patient sample. Centre values represent mean, error bars represent s.d.

    Article Snippet: Immunoblots were performed according to standard procedures using anti-human PARP1 (#9542S) and anti β-actin (#3700S) or anti GAPDH (#5174P) antibodies and HRP-linked secondary reagents (#7074S, anti-rabbit IgG and #7076S, anti-mouse IgG, all from Cell Signaling Technology), and quantified by Fiji software.

    Techniques: Expressing, Inhibition, Quantitative RT-PCR, Derivative Assay, In Vivo, Mouse Assay, In Vitro, Cell Culture, Flow Cytometry, MANN-WHITNEY

    Treatment with a PARP1 inhibitor suppresses in vivo induction of leukaemia in mice co-transplanted with NK cells, but does not affect expression of NKG2DLs in healthy haematopoietic cells and baseline haematopoiesis. a , b , Treatment with AG-14361 in vitro (pre-transplantation) or in vivo (post-transplantation) inhibits the capacity for the in vivo initiation of leukaemia of human AML cells in NSG mice co-transplanted with NK cells. a , In vitro pre-treatment: AML cells were cultured in vitro with AG-14361 (20 μM) or DMSO (0.2%) for 24 h and then transplanted via tail vein injection into NSG mice (1.5 × 10 6 AML cells per mouse), which afterwards were co-transplanted (or not) with pNKCs (1.5 × 10 7 pNKCs per mouse) ( n = 3 mice per condition and patient, n = 5 cases of AML). Mice were analysed for the presence of leukaemic cells in bone marrow, peripheral blood, liver and spleen at 16 h after transplantation. Summarized results of n = 5 independent biological experiments are shown, after normalization to ‘DMSO control without NK cells’ (which was set to 1). Statistical analysis was performed using a two-sided Mann–Whitney U test. b , In vivo treatment: mice transplanted with human AML cells (no. 35, 1.5 × 10 6 AML cells per mouse, transplanted intrafemorally, n = 6 mice per group) were treated with AG-14361 (10 mg kg -1 intraperitoneally, on days 1 to 5 after transplantation) or DMSO control, and afterwards co-transplanted (or not) with pNKCs (4.5 × 10 6 pNKCs per mouse administered once intravenously on day 6 after transplantation, pre-treated or not with anti-NKG2D or isotype control, 5 μg ml -1 ). Mice were analysed at week nine after transplantation for leukaemic engraftment. Note that between day 6 after transplantation and the final analysis time point, no further treatment was applied. See schematic of the experiment and further data with higher (10:1) pNKC:AML cell ratio in Fig. 4j, k. Statistical analyses were performed using a two-sided Student’s t -test. c – i , Surface expression of NKG2DLs on healthy human ( c – f ) and mouse ( g – i ) haematopoietic cells at baseline and after PARP1 inhibition. Representative flow cytometry data of the indicated human haematopoietic cells derived from peripheral blood ( c ) and bone marrow and cord blood ( d – f ) of healthy donors, and quantification ( f ). In g , expression of NKG2DLs is quantified on different subpopulations of healthy mouse haematopoietic bone marrow cells ( n = 3 mice; bulk leukaemic cells from Mll-Enl and Mll-ptd/Flt3-ITD mice are shown as positive controls). h – j , Treatment of mice with AG-14361 (10 mg kg -1 , intraperitoneally) or DMSO vehicle control, and subsequent quantification of expression of NKG2DLs on haematopoietic cells. Schematic of the experiment ( h ) and quantification of percentages of NKG2DL + cells among total mouse haematopoietic cells of specific compartments ( i ) (haematopoietic stem cells (HSCs): KIT + Lin − SCA1 + CD150 + CD48; MEP, megakaryocyte–erythroid progenitor; CLP, common lymphoid progenitor; GMP, granulocyte–macrophage progenitor) and absolute numbers of white blood cell counts ( j ) (with distribution of neutrophils, lymphocytes and monocytes) and red blood cell counts ( n = 3 mice per group). Centre values represent mean, error bars represent s.d. Two-sided statistical tests were performed using Mann–Whitney U test ( a ) or Student’s t -test ( b , f – j ). Centre values represent mean, error bars represent s.d. for all plots.

    Journal: Nature

    Article Title: Absence of NKG2D ligands defines leukaemia stem cells and mediates their immune evasion

    doi: 10.1038/s41586-019-1410-1

    Figure Lengend Snippet: Treatment with a PARP1 inhibitor suppresses in vivo induction of leukaemia in mice co-transplanted with NK cells, but does not affect expression of NKG2DLs in healthy haematopoietic cells and baseline haematopoiesis. a , b , Treatment with AG-14361 in vitro (pre-transplantation) or in vivo (post-transplantation) inhibits the capacity for the in vivo initiation of leukaemia of human AML cells in NSG mice co-transplanted with NK cells. a , In vitro pre-treatment: AML cells were cultured in vitro with AG-14361 (20 μM) or DMSO (0.2%) for 24 h and then transplanted via tail vein injection into NSG mice (1.5 × 10 6 AML cells per mouse), which afterwards were co-transplanted (or not) with pNKCs (1.5 × 10 7 pNKCs per mouse) ( n = 3 mice per condition and patient, n = 5 cases of AML). Mice were analysed for the presence of leukaemic cells in bone marrow, peripheral blood, liver and spleen at 16 h after transplantation. Summarized results of n = 5 independent biological experiments are shown, after normalization to ‘DMSO control without NK cells’ (which was set to 1). Statistical analysis was performed using a two-sided Mann–Whitney U test. b , In vivo treatment: mice transplanted with human AML cells (no. 35, 1.5 × 10 6 AML cells per mouse, transplanted intrafemorally, n = 6 mice per group) were treated with AG-14361 (10 mg kg -1 intraperitoneally, on days 1 to 5 after transplantation) or DMSO control, and afterwards co-transplanted (or not) with pNKCs (4.5 × 10 6 pNKCs per mouse administered once intravenously on day 6 after transplantation, pre-treated or not with anti-NKG2D or isotype control, 5 μg ml -1 ). Mice were analysed at week nine after transplantation for leukaemic engraftment. Note that between day 6 after transplantation and the final analysis time point, no further treatment was applied. See schematic of the experiment and further data with higher (10:1) pNKC:AML cell ratio in Fig. 4j, k. Statistical analyses were performed using a two-sided Student’s t -test. c – i , Surface expression of NKG2DLs on healthy human ( c – f ) and mouse ( g – i ) haematopoietic cells at baseline and after PARP1 inhibition. Representative flow cytometry data of the indicated human haematopoietic cells derived from peripheral blood ( c ) and bone marrow and cord blood ( d – f ) of healthy donors, and quantification ( f ). In g , expression of NKG2DLs is quantified on different subpopulations of healthy mouse haematopoietic bone marrow cells ( n = 3 mice; bulk leukaemic cells from Mll-Enl and Mll-ptd/Flt3-ITD mice are shown as positive controls). h – j , Treatment of mice with AG-14361 (10 mg kg -1 , intraperitoneally) or DMSO vehicle control, and subsequent quantification of expression of NKG2DLs on haematopoietic cells. Schematic of the experiment ( h ) and quantification of percentages of NKG2DL + cells among total mouse haematopoietic cells of specific compartments ( i ) (haematopoietic stem cells (HSCs): KIT + Lin − SCA1 + CD150 + CD48; MEP, megakaryocyte–erythroid progenitor; CLP, common lymphoid progenitor; GMP, granulocyte–macrophage progenitor) and absolute numbers of white blood cell counts ( j ) (with distribution of neutrophils, lymphocytes and monocytes) and red blood cell counts ( n = 3 mice per group). Centre values represent mean, error bars represent s.d. Two-sided statistical tests were performed using Mann–Whitney U test ( a ) or Student’s t -test ( b , f – j ). Centre values represent mean, error bars represent s.d. for all plots.

    Article Snippet: Immunoblots were performed according to standard procedures using anti-human PARP1 (#9542S) and anti β-actin (#3700S) or anti GAPDH (#5174P) antibodies and HRP-linked secondary reagents (#7074S, anti-rabbit IgG and #7076S, anti-mouse IgG, all from Cell Signaling Technology), and quantified by Fiji software.

    Techniques: In Vivo, Mouse Assay, Expressing, In Vitro, Transplantation Assay, Cell Culture, Injection, MANN-WHITNEY, Inhibition, Flow Cytometry, Derivative Assay

    Detection of apoptosis in retinas of rbpr2 fs-muz99 mutants. ( A ) Wild-type (WT) and rbpr2 mutant ( rbpr2 fs-muz99 ) zebrafish retinas at 5.5 dpf were stained with TUNEL reagent. The TUNEL positive cells/apoptotic nuclei stain green. ( B ) Quantification of TUNEL positive cells. ( C ) Determination of rod and cone opsin levels in WT and mutant eyes (dissected heads; n = 12 each genotype) by Western blot analysis at 8 dpf. ( D ) Total protein from WT and mutant eyes (dissected heads; n = 12 each genotype), at the 5.5 and 8 dpf time-points, were pooled and subjected to Western blot analysis using PARP1 antibody. Cleaved PARP1 products in rbpr2 fs-muz99 mutant eyes indicate apoptosis. TUNEL assays and Western blot experiments were repeated thrice. For Figure 9 B, statistical analysis of data using the Mann–Whitney U test showed a p

    Journal: Cells

    Article Title: A Functional Binding Domain in the Rbpr2 Receptor Is Required for Vitamin A Transport, Ocular Retinoid Homeostasis, and Photoreceptor Cell Survival in Zebrafish

    doi: 10.3390/cells9051099

    Figure Lengend Snippet: Detection of apoptosis in retinas of rbpr2 fs-muz99 mutants. ( A ) Wild-type (WT) and rbpr2 mutant ( rbpr2 fs-muz99 ) zebrafish retinas at 5.5 dpf were stained with TUNEL reagent. The TUNEL positive cells/apoptotic nuclei stain green. ( B ) Quantification of TUNEL positive cells. ( C ) Determination of rod and cone opsin levels in WT and mutant eyes (dissected heads; n = 12 each genotype) by Western blot analysis at 8 dpf. ( D ) Total protein from WT and mutant eyes (dissected heads; n = 12 each genotype), at the 5.5 and 8 dpf time-points, were pooled and subjected to Western blot analysis using PARP1 antibody. Cleaved PARP1 products in rbpr2 fs-muz99 mutant eyes indicate apoptosis. TUNEL assays and Western blot experiments were repeated thrice. For Figure 9 B, statistical analysis of data using the Mann–Whitney U test showed a p

    Article Snippet: Membranes were probed with primary antibodies against β-Actin or Gapdh (1:10,000, Sigma/Millipore, Cambridge, MA, USA), anti-V5 (1:1000, Sigma/Millipore, Cambridge, MA, USA), anti-Lrat (1:1000; Abcam, Cambridge, MA, USA), anti-Rhodopsin (1:1000, Sigma/Millipore, Cambridge, MA, USA), anti-PARP1 (1:2000; Cell Signaling, Danvers, MA) and anti-Red/Green cone opsin (1:1000, Sigma/Millipore, Cambridge, MA, USA) in antibody buffer (0.2% Triton X-100, 2% BSA, 1X PBS) [ , , , ].

    Techniques: Mutagenesis, Staining, TUNEL Assay, Western Blot, MANN-WHITNEY

    PARylation of Histone H3 decreases EZH2-mediated histone methylation (A) Schematic of experimental approach that couples histone PARylation and methylation in vitro . First, histone H3 was incubated with PARP1 in the presence or absence of NAD+ and olaparib to allow for PARylation. After 60 minutes, the reaction was blocked by addition of olaparib, PARP1 was removed by immunoprecipitation and the remaining histone H3 was either assessed for PARylation by PAR-resin pulldown or incubated with EZH2/PRC2 and SAM to allow H3-K27 methylation in vitro . After 30 minutes, the histone methyltransferase reaction was blocked and H3K27me3 levels were determined by different approaches. (B) Histone H3 PARylation decreases subsequent H3-K27 methylation. Histone H3 proteins treated as in A) were analyzed by western blot using an anti-H3K27me3 antibody and an anti-histone H3 antibody as a control. The signal intensity of H3K27me3 relative to H3 was measured using ImageJ software and normalized to the signal from unmodified histone H3 (H3 incubated with PARP1 in the absence of NAD+, lane 1). PARP1 activity was confirmed by western blot using an anti-PAR antibody. The western blot is representative of three independent experiments. (C) PARylation reduces histone methylation in vitro . Histone H3 samples treated as in A) were used to determine EZH2 activity toward unmodified and PARylated histone H3 by measuring H3K27me3 levels using an ELISA kit. H3K27me3 levels from unmodified histone H3 were set as 100% EZH2 activity. N=3, mean ± SD. (D) In vitro PARylation of histone H3. Histone H3 proteins treated as in A) were immunoprecipitated using the PAR-affinity resin and PARylation of H3 was confirmed by western blot using an anti-H3 antibody. PAR-resin specificity and PARylation levels were determined by western blot analysis of purified proteins with an anti-PAR antibody. The smear observed in lane 2 indicated PARylation. H. (E) PARP1 Immunoprecipitation. PARP1 removal after PARylation of histone H3 treated as above was confirmed by western blot analysis of proteins immunoprecipitated with an anti-PARP1 antibody.

    Journal: Oncotarget

    Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

    doi: 10.18632/oncotarget.24291

    Figure Lengend Snippet: PARylation of Histone H3 decreases EZH2-mediated histone methylation (A) Schematic of experimental approach that couples histone PARylation and methylation in vitro . First, histone H3 was incubated with PARP1 in the presence or absence of NAD+ and olaparib to allow for PARylation. After 60 minutes, the reaction was blocked by addition of olaparib, PARP1 was removed by immunoprecipitation and the remaining histone H3 was either assessed for PARylation by PAR-resin pulldown or incubated with EZH2/PRC2 and SAM to allow H3-K27 methylation in vitro . After 30 minutes, the histone methyltransferase reaction was blocked and H3K27me3 levels were determined by different approaches. (B) Histone H3 PARylation decreases subsequent H3-K27 methylation. Histone H3 proteins treated as in A) were analyzed by western blot using an anti-H3K27me3 antibody and an anti-histone H3 antibody as a control. The signal intensity of H3K27me3 relative to H3 was measured using ImageJ software and normalized to the signal from unmodified histone H3 (H3 incubated with PARP1 in the absence of NAD+, lane 1). PARP1 activity was confirmed by western blot using an anti-PAR antibody. The western blot is representative of three independent experiments. (C) PARylation reduces histone methylation in vitro . Histone H3 samples treated as in A) were used to determine EZH2 activity toward unmodified and PARylated histone H3 by measuring H3K27me3 levels using an ELISA kit. H3K27me3 levels from unmodified histone H3 were set as 100% EZH2 activity. N=3, mean ± SD. (D) In vitro PARylation of histone H3. Histone H3 proteins treated as in A) were immunoprecipitated using the PAR-affinity resin and PARylation of H3 was confirmed by western blot using an anti-H3 antibody. PAR-resin specificity and PARylation levels were determined by western blot analysis of purified proteins with an anti-PAR antibody. The smear observed in lane 2 indicated PARylation. H. (E) PARP1 Immunoprecipitation. PARP1 removal after PARylation of histone H3 treated as above was confirmed by western blot analysis of proteins immunoprecipitated with an anti-PARP1 antibody.

    Article Snippet: In vitro PARylation assay Recombinant proteins (500 ng) were incubated in 20 mL PAR reaction buffer containing 0.5 U of human PARP1 enzyme (Tulip, #2090), 50 μM Tris (pH 8.0), 10 μM MgCl2 , 5 μM KCl, with or without 1 μM NAD+ , and with or without 1 μg/mL activated DNA (Sigma D4522).

    Techniques: Methylation, In Vitro, Incubation, Immunoprecipitation, Western Blot, Software, Activity Assay, Enzyme-linked Immunosorbent Assay, Purification

    EZH2 PARylation decreases global H3K27me3 levels after DNA damage (A) PARylation of EZH2 after DNA damage by UV radiation. HeLa cells were irradiated with UVA and UVB for 2 minutes and then recovered in media. PARylated proteins were pulled down at the indicated recovery times using PAR-resin and analyzed by western blot using anti-PARP1, anti-EZH2 and anti-PAR antibodies. The shift of EZH2 to a higher molecular weight isoform indicates its PARylation. EZH2 PARylation increases and reaches a maximum at 3h after the initial treatment with UV. Input corresponds to 1/20th the amount of protein extract. (B and C) HeLa cells were treated as above and proteins were pulled down using either an anti mono-ADP-ribose antibody or an anti mono/poly-ADP-ribose antibody. Immunoprecipitated proteins were then analyzed by western blot with anti-EZH2 and anti-PARP1 antibodies. The western blot is representative of at least three independent experiments. Untreated cells served as control. (D) Histones were extracted from HeLa cells treated as described above. H3K27me3 levels were analyzed by western blot with an anti-H3K27me3 antibody or anti-histone H3 as control. (E) Quantification of H3K27me3 levels after UV irradiation-induced DNA damage. HeLa cells were exposed to UV as described in A) and recovered in media. After 3 hours, histones were purified and assessed for H3K27me3 levels by ELISA. The level of K27 methylation under the indicated conditions were calculated based on the amount of H3-K27 converted in the assay, divided by the amount of total histones loaded. Data were normalized to the untreated and expressed as % of H3K27me3. Data were N=3, mean ± SD. Statistically significant differences between experimental conditions and control samples were determined by Student's t test.

    Journal: Oncotarget

    Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

    doi: 10.18632/oncotarget.24291

    Figure Lengend Snippet: EZH2 PARylation decreases global H3K27me3 levels after DNA damage (A) PARylation of EZH2 after DNA damage by UV radiation. HeLa cells were irradiated with UVA and UVB for 2 minutes and then recovered in media. PARylated proteins were pulled down at the indicated recovery times using PAR-resin and analyzed by western blot using anti-PARP1, anti-EZH2 and anti-PAR antibodies. The shift of EZH2 to a higher molecular weight isoform indicates its PARylation. EZH2 PARylation increases and reaches a maximum at 3h after the initial treatment with UV. Input corresponds to 1/20th the amount of protein extract. (B and C) HeLa cells were treated as above and proteins were pulled down using either an anti mono-ADP-ribose antibody or an anti mono/poly-ADP-ribose antibody. Immunoprecipitated proteins were then analyzed by western blot with anti-EZH2 and anti-PARP1 antibodies. The western blot is representative of at least three independent experiments. Untreated cells served as control. (D) Histones were extracted from HeLa cells treated as described above. H3K27me3 levels were analyzed by western blot with an anti-H3K27me3 antibody or anti-histone H3 as control. (E) Quantification of H3K27me3 levels after UV irradiation-induced DNA damage. HeLa cells were exposed to UV as described in A) and recovered in media. After 3 hours, histones were purified and assessed for H3K27me3 levels by ELISA. The level of K27 methylation under the indicated conditions were calculated based on the amount of H3-K27 converted in the assay, divided by the amount of total histones loaded. Data were normalized to the untreated and expressed as % of H3K27me3. Data were N=3, mean ± SD. Statistically significant differences between experimental conditions and control samples were determined by Student's t test.

    Article Snippet: In vitro PARylation assay Recombinant proteins (500 ng) were incubated in 20 mL PAR reaction buffer containing 0.5 U of human PARP1 enzyme (Tulip, #2090), 50 μM Tris (pH 8.0), 10 μM MgCl2 , 5 μM KCl, with or without 1 μM NAD+ , and with or without 1 μg/mL activated DNA (Sigma D4522).

    Techniques: Irradiation, Western Blot, Molecular Weight, Immunoprecipitation, Purification, Enzyme-linked Immunosorbent Assay, Methylation

    EZH2 interaction with chromatin decreases after DNA damage (A and B) EZH2 association with chromatin after DNA damage. HEK293 cells and HeLa cells were treated with or without 100 uM MNNG to induce DNA damage. After 10 minutes, proteins were extracted and fractionated to obtain nuclear soluble and chromatin-bound protein extracts. The fractionated proteins were analyzed for EZH2 expression by western blot using an anti-EZH2 antibody. The effectiveness of separation of nuclear soluble and chromatin-bound proteins was determined by western blot with an anti-histone H3 antibody. All western blots are representative of at least three independent experiments. (C) Global levels of nuclear soluble proteins and chromatin-bound proteins are unaffected by PARP1 activation. Nuclear soluble and chromatin-bound protein extracts from HEK293 cells and HeLa cells treated as in B) were separated by gel electrophoresis on a 4-20% polyacrylamide gel and stained with coomassie brilliant blue to confirm both correct fraction separation and equal protein quantity. The image is representative of at least three independent experiments.

    Journal: Oncotarget

    Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

    doi: 10.18632/oncotarget.24291

    Figure Lengend Snippet: EZH2 interaction with chromatin decreases after DNA damage (A and B) EZH2 association with chromatin after DNA damage. HEK293 cells and HeLa cells were treated with or without 100 uM MNNG to induce DNA damage. After 10 minutes, proteins were extracted and fractionated to obtain nuclear soluble and chromatin-bound protein extracts. The fractionated proteins were analyzed for EZH2 expression by western blot using an anti-EZH2 antibody. The effectiveness of separation of nuclear soluble and chromatin-bound proteins was determined by western blot with an anti-histone H3 antibody. All western blots are representative of at least three independent experiments. (C) Global levels of nuclear soluble proteins and chromatin-bound proteins are unaffected by PARP1 activation. Nuclear soluble and chromatin-bound protein extracts from HEK293 cells and HeLa cells treated as in B) were separated by gel electrophoresis on a 4-20% polyacrylamide gel and stained with coomassie brilliant blue to confirm both correct fraction separation and equal protein quantity. The image is representative of at least three independent experiments.

    Article Snippet: In vitro PARylation assay Recombinant proteins (500 ng) were incubated in 20 mL PAR reaction buffer containing 0.5 U of human PARP1 enzyme (Tulip, #2090), 50 μM Tris (pH 8.0), 10 μM MgCl2 , 5 μM KCl, with or without 1 μM NAD+ , and with or without 1 μg/mL activated DNA (Sigma D4522).

    Techniques: Expressing, Western Blot, Activation Assay, Nucleic Acid Electrophoresis, Staining

    EZH2 inhibitor UNC1999 enhanced PARP1 inhibitor olaparib-mediated synthetic lethality in BRCA-deficient cell lines and acute myeloid leukemia (AML) primary cells (A) BRCA1 -mutated and BRCA1 -reconstituted MDA-MB-436 human breast carcinoma cells were treated with or without the PARP1 inhibitor olaparib in the presence or absence of the EZH2 inhibitor UNC1999 at the indicated concentrations. After 4 days, cell count/viability was determined by Trypan blue exclusion using a Bio Rad TC20 Automated Cell Counter. Results show mean ± SD of living cells. N=3. * p

    Journal: Oncotarget

    Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

    doi: 10.18632/oncotarget.24291

    Figure Lengend Snippet: EZH2 inhibitor UNC1999 enhanced PARP1 inhibitor olaparib-mediated synthetic lethality in BRCA-deficient cell lines and acute myeloid leukemia (AML) primary cells (A) BRCA1 -mutated and BRCA1 -reconstituted MDA-MB-436 human breast carcinoma cells were treated with or without the PARP1 inhibitor olaparib in the presence or absence of the EZH2 inhibitor UNC1999 at the indicated concentrations. After 4 days, cell count/viability was determined by Trypan blue exclusion using a Bio Rad TC20 Automated Cell Counter. Results show mean ± SD of living cells. N=3. * p

    Article Snippet: In vitro PARylation assay Recombinant proteins (500 ng) were incubated in 20 mL PAR reaction buffer containing 0.5 U of human PARP1 enzyme (Tulip, #2090), 50 μM Tris (pH 8.0), 10 μM MgCl2 , 5 μM KCl, with or without 1 μM NAD+ , and with or without 1 μg/mL activated DNA (Sigma D4522).

    Techniques: Multiple Displacement Amplification, Cell Counting

    PARP1 PARylates EZH2 and inhibits EZH2 activity in vitro (A) PARylation of EZH2 by PARP1 in vitro . Human EZH2/PRC2 complex (EZH2, EED, SUZ12, RbAP48 and AEBP2) was incubated alone (lane 1) or with the agents indicated at the top (250 nM of olaparib (PARP inhibitor) was used and NAD+ is necessary for PARP1 activity). After 1 hour, PARylation was blocked by adding olaparib to all samples and PARylated proteins were pulled-down by PAR-affinity resin and analyzed by western blot with anti-EZH2 and anti-PAR antibodies. PARylation appears as a smear due to the different sizes of the various PAR polymers. Input corresponds to 1/10 th the amount of protein used for immunoprecipitation. Input was immunoblotted with anti-PARP1 and anti-EZH2 antibodies. (B) Schematic of the experimental strategy for C) and D). Briefly, the EZH2/PRC2 complex was incubated with PARP1 in the presence or absence of NAD+ as in A). After 1 hour, PARylation was stopped with olaparib and PARP1 was removed by immunoprecipitation with an anti-PARP1 antibody. The EZH2/PRC2 complex was incubated with EZH2 substrates histone H3 and S-adenosyl methionine (SAM) to allow histone methylation to occur. After 30 minutes, histone methytransferase activity was determined by assessing H3K27me3 levels. (C) In vitro histone methyltransferase assay. As indicated in B), purified histone H3 and SAM were incubated with the agents indicated at the top. After 30 minutes, proteins were analyzed by western blot using anti-Histone H3, anti-H3K27me3 and anti-PAR antibodies. Input corresponds to 1/20 th the amount of the protein used for immunoblotting. Input was probed with an anti-EZH2 antibody. (D) Levels of EZH2 activity with (black) and without (grey) PARP1 activity. Extracts from the EZH2/PRC2 complex incubated with histone H3 and SAM as in lanes 2 and 4 from C) were assessed for H3K27me3 levels by ELISA. N=3 ± SD.

    Journal: Oncotarget

    Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

    doi: 10.18632/oncotarget.24291

    Figure Lengend Snippet: PARP1 PARylates EZH2 and inhibits EZH2 activity in vitro (A) PARylation of EZH2 by PARP1 in vitro . Human EZH2/PRC2 complex (EZH2, EED, SUZ12, RbAP48 and AEBP2) was incubated alone (lane 1) or with the agents indicated at the top (250 nM of olaparib (PARP inhibitor) was used and NAD+ is necessary for PARP1 activity). After 1 hour, PARylation was blocked by adding olaparib to all samples and PARylated proteins were pulled-down by PAR-affinity resin and analyzed by western blot with anti-EZH2 and anti-PAR antibodies. PARylation appears as a smear due to the different sizes of the various PAR polymers. Input corresponds to 1/10 th the amount of protein used for immunoprecipitation. Input was immunoblotted with anti-PARP1 and anti-EZH2 antibodies. (B) Schematic of the experimental strategy for C) and D). Briefly, the EZH2/PRC2 complex was incubated with PARP1 in the presence or absence of NAD+ as in A). After 1 hour, PARylation was stopped with olaparib and PARP1 was removed by immunoprecipitation with an anti-PARP1 antibody. The EZH2/PRC2 complex was incubated with EZH2 substrates histone H3 and S-adenosyl methionine (SAM) to allow histone methylation to occur. After 30 minutes, histone methytransferase activity was determined by assessing H3K27me3 levels. (C) In vitro histone methyltransferase assay. As indicated in B), purified histone H3 and SAM were incubated with the agents indicated at the top. After 30 minutes, proteins were analyzed by western blot using anti-Histone H3, anti-H3K27me3 and anti-PAR antibodies. Input corresponds to 1/20 th the amount of the protein used for immunoblotting. Input was probed with an anti-EZH2 antibody. (D) Levels of EZH2 activity with (black) and without (grey) PARP1 activity. Extracts from the EZH2/PRC2 complex incubated with histone H3 and SAM as in lanes 2 and 4 from C) were assessed for H3K27me3 levels by ELISA. N=3 ± SD.

    Article Snippet: In vitro PARylation assay Recombinant proteins (500 ng) were incubated in 20 mL PAR reaction buffer containing 0.5 U of human PARP1 enzyme (Tulip, #2090), 50 μM Tris (pH 8.0), 10 μM MgCl2 , 5 μM KCl, with or without 1 μM NAD+ , and with or without 1 μg/mL activated DNA (Sigma D4522).

    Techniques: Activity Assay, In Vitro, Incubation, Western Blot, Immunoprecipitation, Methylation, HMT Assay, Purification, Enzyme-linked Immunosorbent Assay

    PARylation of EZH2 stably inhibits EZH2 enzymatic activity (A) Time course of in vitro PARylation assay. EZH2/PRC2 complex was incubated with PARP1, NAD+ and DNA fragments to allow in vitro PARylation. The reaction was blocked at different time points by adding the PARP inhibitor olaparib. After removing PARP1 from the reaction, PARylated proteins were pulled down with a PAR-affinity resin and analyzed by western blot with an anti-EZH2 antibody. Input corresponds to 1/10 th the amount of protein used for PAR pulldown. Input was probed with an anti-EZH2 antibody. (B) In vitro histone methylation assay. EZH2/PRC2 complex treated as in A) was incubated with histone H3 and SAM to allow methylation of lysine 27 of histone H3. After 30 minutes, histone H3 was extracted and H3K27me3 levels were measured by ELISA. EZH2 activity was calculated by setting H3K27me3 levels at time 0 as 100% EZH2 activity. N=3 mean ± SD. (C) In vitro histone methyltransferase activity assay. EZH2/PRC2 complex was incubated with PARP1 in the presence (PARylated) or absence (unmodified) of NAD+. After 1 hour, the reaction was blocked as in A) and EZH2/PRC2 complex was incubated with SAM and different concentrations of histone H3 to allow histone H3-K27 methylation to occur. After 30 minutes, the reaction was blocked and the amount of methylated histone H3-K27 generated by EZH2 activity was measured using an H3K27me3 ELISA kit. N=3, mean ± SD. (D) Time course of in vitro histone methyltransferase (HMT) activity. EZH2/PRC2 complex was treated as in C) and incubated with SAM and histone H3 to allow methylation of H3-K27. The reaction was blocked at different time points and the amount of methylated histone H3-K27 generated by EZH2 activity was measured by an H3K27me3 ELISA. N=3, mean ± SD.

    Journal: Oncotarget

    Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

    doi: 10.18632/oncotarget.24291

    Figure Lengend Snippet: PARylation of EZH2 stably inhibits EZH2 enzymatic activity (A) Time course of in vitro PARylation assay. EZH2/PRC2 complex was incubated with PARP1, NAD+ and DNA fragments to allow in vitro PARylation. The reaction was blocked at different time points by adding the PARP inhibitor olaparib. After removing PARP1 from the reaction, PARylated proteins were pulled down with a PAR-affinity resin and analyzed by western blot with an anti-EZH2 antibody. Input corresponds to 1/10 th the amount of protein used for PAR pulldown. Input was probed with an anti-EZH2 antibody. (B) In vitro histone methylation assay. EZH2/PRC2 complex treated as in A) was incubated with histone H3 and SAM to allow methylation of lysine 27 of histone H3. After 30 minutes, histone H3 was extracted and H3K27me3 levels were measured by ELISA. EZH2 activity was calculated by setting H3K27me3 levels at time 0 as 100% EZH2 activity. N=3 mean ± SD. (C) In vitro histone methyltransferase activity assay. EZH2/PRC2 complex was incubated with PARP1 in the presence (PARylated) or absence (unmodified) of NAD+. After 1 hour, the reaction was blocked as in A) and EZH2/PRC2 complex was incubated with SAM and different concentrations of histone H3 to allow histone H3-K27 methylation to occur. After 30 minutes, the reaction was blocked and the amount of methylated histone H3-K27 generated by EZH2 activity was measured using an H3K27me3 ELISA kit. N=3, mean ± SD. (D) Time course of in vitro histone methyltransferase (HMT) activity. EZH2/PRC2 complex was treated as in C) and incubated with SAM and histone H3 to allow methylation of H3-K27. The reaction was blocked at different time points and the amount of methylated histone H3-K27 generated by EZH2 activity was measured by an H3K27me3 ELISA. N=3, mean ± SD.

    Article Snippet: In vitro PARylation assay Recombinant proteins (500 ng) were incubated in 20 mL PAR reaction buffer containing 0.5 U of human PARP1 enzyme (Tulip, #2090), 50 μM Tris (pH 8.0), 10 μM MgCl2 , 5 μM KCl, with or without 1 μM NAD+ , and with or without 1 μg/mL activated DNA (Sigma D4522).

    Techniques: Stable Transfection, Activity Assay, In Vitro, Incubation, Western Blot, Methylation, Enzyme-linked Immunosorbent Assay, Generated, HMT Assay

    PARG reverses EZH2 PARylation and restores EZH2 enzymatic activity (A) In vitro PARG assay. EZH2/PRC2 complex and PARP1 were incubated with or without PARG as indicated (Note: NAD+ is required for PARylation). After 1 hour, the reaction was blocked by addition of the PARP inhibitor olaparib and the EZH2/PRC2 complex was incubated with (lanes 2 and 4) or without (lanes 1 and 3) PARG to allow degradation of PAR polymers. After 1 hour, the reaction was stopped by adding Laemmli buffer and the proteins were analyzed by western blot using anti-EZH2 and anti-PAR antibodies. The upper band in the top panel represents PARylated EZH2. PARG activity was confirmed by reduction of PAR smear. (B) In vitro histone methyltransferase (HMT) activity assay. EZH2/PRC2 complex treated as in A) was subsequently assayed for histone methyltransferase activity using an HMT assay kit. The activity of EZH2 under the indicated conditions was calculated based on the amount of H3-K27 converted in the assay. As a control, the activity of EZH2/PRC2 complex alone was also determined. N=3, mean ± SD.

    Journal: Oncotarget

    Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

    doi: 10.18632/oncotarget.24291

    Figure Lengend Snippet: PARG reverses EZH2 PARylation and restores EZH2 enzymatic activity (A) In vitro PARG assay. EZH2/PRC2 complex and PARP1 were incubated with or without PARG as indicated (Note: NAD+ is required for PARylation). After 1 hour, the reaction was blocked by addition of the PARP inhibitor olaparib and the EZH2/PRC2 complex was incubated with (lanes 2 and 4) or without (lanes 1 and 3) PARG to allow degradation of PAR polymers. After 1 hour, the reaction was stopped by adding Laemmli buffer and the proteins were analyzed by western blot using anti-EZH2 and anti-PAR antibodies. The upper band in the top panel represents PARylated EZH2. PARG activity was confirmed by reduction of PAR smear. (B) In vitro histone methyltransferase (HMT) activity assay. EZH2/PRC2 complex treated as in A) was subsequently assayed for histone methyltransferase activity using an HMT assay kit. The activity of EZH2 under the indicated conditions was calculated based on the amount of H3-K27 converted in the assay. As a control, the activity of EZH2/PRC2 complex alone was also determined. N=3, mean ± SD.

    Article Snippet: In vitro PARylation assay Recombinant proteins (500 ng) were incubated in 20 mL PAR reaction buffer containing 0.5 U of human PARP1 enzyme (Tulip, #2090), 50 μM Tris (pH 8.0), 10 μM MgCl2 , 5 μM KCl, with or without 1 μM NAD+ , and with or without 1 μg/mL activated DNA (Sigma D4522).

    Techniques: Activity Assay, In Vitro, PARG Assay, Incubation, Western Blot, HMT Assay

    PARylation of histone H3 decreases EZH2 affinity for H3, while methylation of histone H3 has no effect on the ability of PARP1 to interact with histone H3 (A) Schematic of histone peptide pull-down after PARylation. (B) Histone peptide pull-down for EZH2. Synthesized histone H3 peptide, corresponding to residues 21-44 of human histone H3, was conjugated with biotin and incubated with PARP1 in the presence or absence of NAD+ to allow for PARylation. After 1 hour, the reaction was blocked with 250 nM olaparib and the H3 peptide was immunopurified with streptavidin-magnetic beads and subsequently incubated with EZH2/PRC2 complex. After 4 hours, the peptide-coated, streptavidin-conjugated beads were washed to remove unbound proteins and bound proteins were analyzed by western blot using an anti-EZH2 antibody. PARylation of the peptide was confirmed by western blot using an anti-PAR antibody. Top panel shows short film exposure; lower panel longer film exposure. (C) Schematic of histone peptide pull-down after in vitro histone methyltransferase assay. (D) Histone peptide pull-down assay for PARP1. Synthesized histone H3 peptide containing tri-methylated lysine 27 was conjugated with streptavidin magnetic beads followed by incubation with purified PARP1. After 4 hours, the peptide-coated, streptavidin-conjugated beads were washed to remove unbound proteins and bound proteins were analyzed by western blot using an anti-PARP1 antibody.

    Journal: Oncotarget

    Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

    doi: 10.18632/oncotarget.24291

    Figure Lengend Snippet: PARylation of histone H3 decreases EZH2 affinity for H3, while methylation of histone H3 has no effect on the ability of PARP1 to interact with histone H3 (A) Schematic of histone peptide pull-down after PARylation. (B) Histone peptide pull-down for EZH2. Synthesized histone H3 peptide, corresponding to residues 21-44 of human histone H3, was conjugated with biotin and incubated with PARP1 in the presence or absence of NAD+ to allow for PARylation. After 1 hour, the reaction was blocked with 250 nM olaparib and the H3 peptide was immunopurified with streptavidin-magnetic beads and subsequently incubated with EZH2/PRC2 complex. After 4 hours, the peptide-coated, streptavidin-conjugated beads were washed to remove unbound proteins and bound proteins were analyzed by western blot using an anti-EZH2 antibody. PARylation of the peptide was confirmed by western blot using an anti-PAR antibody. Top panel shows short film exposure; lower panel longer film exposure. (C) Schematic of histone peptide pull-down after in vitro histone methyltransferase assay. (D) Histone peptide pull-down assay for PARP1. Synthesized histone H3 peptide containing tri-methylated lysine 27 was conjugated with streptavidin magnetic beads followed by incubation with purified PARP1. After 4 hours, the peptide-coated, streptavidin-conjugated beads were washed to remove unbound proteins and bound proteins were analyzed by western blot using an anti-PARP1 antibody.

    Article Snippet: In vitro PARylation assay Recombinant proteins (500 ng) were incubated in 20 mL PAR reaction buffer containing 0.5 U of human PARP1 enzyme (Tulip, #2090), 50 μM Tris (pH 8.0), 10 μM MgCl2 , 5 μM KCl, with or without 1 μM NAD+ , and with or without 1 μg/mL activated DNA (Sigma D4522).

    Techniques: Methylation, Synthesized, Incubation, Magnetic Beads, Western Blot, In Vitro, HMT Assay, Pull Down Assay, Purification

    EZH2 interacts with PARP1 and is PARylated after DNA damage induction (A) Immunoprecipitation of EZH2 with PARP1 under physiological conditions and after induction of DNA damage. His-PARP1 was expressed in HeLa cells by transfection. Cells were treated with 100 uM of the alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) for 10 minutes to induce DNA damage and activate PARP1. Input corresponds to 1/20 th of protein extracts from transfected cells used for the tag-construct pulldown. (B) Proteins interacting with EZH2 were analyzed by His pulldown or immunoprecipitated with non-immunogenic IgG (control) followed by western blot analysis with anti-EZH2 (top), anti-PAR (middle) and anti-PARP1 (bottom) antibodies. (C) Immunoprecipitation of EZH2 with PAR-affinity resin after induction of DNA damage. LCLs and HeLa cells were treated with or without 100 uM MNNG for 10 minutes. Cellular protein extracts were immunoprecipitated with a PAR affinity resin or PAR negative control resin and analyzed by western blot with anti-EZH2 and anti-PARP1 antibodies. Input corresponds to 1/10 th the amount of cell extracts used for immunoprecipitation.

    Journal: Oncotarget

    Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

    doi: 10.18632/oncotarget.24291

    Figure Lengend Snippet: EZH2 interacts with PARP1 and is PARylated after DNA damage induction (A) Immunoprecipitation of EZH2 with PARP1 under physiological conditions and after induction of DNA damage. His-PARP1 was expressed in HeLa cells by transfection. Cells were treated with 100 uM of the alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) for 10 minutes to induce DNA damage and activate PARP1. Input corresponds to 1/20 th of protein extracts from transfected cells used for the tag-construct pulldown. (B) Proteins interacting with EZH2 were analyzed by His pulldown or immunoprecipitated with non-immunogenic IgG (control) followed by western blot analysis with anti-EZH2 (top), anti-PAR (middle) and anti-PARP1 (bottom) antibodies. (C) Immunoprecipitation of EZH2 with PAR-affinity resin after induction of DNA damage. LCLs and HeLa cells were treated with or without 100 uM MNNG for 10 minutes. Cellular protein extracts were immunoprecipitated with a PAR affinity resin or PAR negative control resin and analyzed by western blot with anti-EZH2 and anti-PARP1 antibodies. Input corresponds to 1/10 th the amount of cell extracts used for immunoprecipitation.

    Article Snippet: In vitro PARylation assay Recombinant proteins (500 ng) were incubated in 20 mL PAR reaction buffer containing 0.5 U of human PARP1 enzyme (Tulip, #2090), 50 μM Tris (pH 8.0), 10 μM MgCl2 , 5 μM KCl, with or without 1 μM NAD+ , and with or without 1 μg/mL activated DNA (Sigma D4522).

    Techniques: Immunoprecipitation, Transfection, Construct, Western Blot, Negative Control

    RT-PCR of HDACI expression in THP-1 cells treated with LPS in the presence or absence of DIQ (300 μmol/L), a PARP1 inhibitor. THP-1 cells were treated with LPS (10 μg/mL) for 4 or 8 h. Error bars indicate mean ± SEM.

    Journal: Molecular Medicine

    Article Title: Poly(ADP-Ribose) Polymerase 1–Sirtuin 1 Functional Interplay Regulates LPS-Mediated High Mobility Group Box 1 Secretion

    doi: 10.2119/molmed.2014.00156

    Figure Lengend Snippet: RT-PCR of HDACI expression in THP-1 cells treated with LPS in the presence or absence of DIQ (300 μmol/L), a PARP1 inhibitor. THP-1 cells were treated with LPS (10 μg/mL) for 4 or 8 h. Error bars indicate mean ± SEM.

    Article Snippet: In contrast, PARP1−/− macrophages exposed to LPS demonstrated no detectable HMGB1 levels in the supernatant ( ).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

    (A) Nuclear proteins were extracted from THP-1 cells treated with LPS in the presence or absence of DIQ (300 μmol/L) and NAM (5 and 10 mmol/L). THP-1 cells were treated with LPS (10 μg/mL) for 4, 8, 12 h. SIRT1 activity was determined by colorimetric assay. (*Represents P ≤ 0.05 LPS exposed cells in the absence or presence of DIQ; # represents P ≤ 0.05 LPS exposed cells treated with DIQ or DIQ and NAM.) Assay shown is representative of three experiments with similar results. (B) Representative autoradiograph of Western blot analysis for nuclear SIRT1 concentration in THP-1 cells treated with LPS in the presence or absence of DIQ (100 and 300 μmol/L). THP-1 cells were treated with LPS (10 μg/mL) for 4 and 8 h. The gel is representative of three experiments with similar results. (C) Nuclear proteins were extracted from WT and PARP1 −/− cells and SIRT1 activity was determined by colorimetric assay. (*Represents P ≤ 0.05 versus WT cells.) (D) Representative autoradiograph of Western blot analysis for supernatant HMGB1 levels in THP-1 cells treated with LPS in the presence or absence of SRT1720 (0.01 and 0.1 μmol/L), a SIRT1 activator. THP-1 cells were treated with LPS (10 μg/mL) for 18 h. The gel is representative of three experiments with similar results.

    Journal: Molecular Medicine

    Article Title: Poly(ADP-Ribose) Polymerase 1–Sirtuin 1 Functional Interplay Regulates LPS-Mediated High Mobility Group Box 1 Secretion

    doi: 10.2119/molmed.2014.00156

    Figure Lengend Snippet: (A) Nuclear proteins were extracted from THP-1 cells treated with LPS in the presence or absence of DIQ (300 μmol/L) and NAM (5 and 10 mmol/L). THP-1 cells were treated with LPS (10 μg/mL) for 4, 8, 12 h. SIRT1 activity was determined by colorimetric assay. (*Represents P ≤ 0.05 LPS exposed cells in the absence or presence of DIQ; # represents P ≤ 0.05 LPS exposed cells treated with DIQ or DIQ and NAM.) Assay shown is representative of three experiments with similar results. (B) Representative autoradiograph of Western blot analysis for nuclear SIRT1 concentration in THP-1 cells treated with LPS in the presence or absence of DIQ (100 and 300 μmol/L). THP-1 cells were treated with LPS (10 μg/mL) for 4 and 8 h. The gel is representative of three experiments with similar results. (C) Nuclear proteins were extracted from WT and PARP1 −/− cells and SIRT1 activity was determined by colorimetric assay. (*Represents P ≤ 0.05 versus WT cells.) (D) Representative autoradiograph of Western blot analysis for supernatant HMGB1 levels in THP-1 cells treated with LPS in the presence or absence of SRT1720 (0.01 and 0.1 μmol/L), a SIRT1 activator. THP-1 cells were treated with LPS (10 μg/mL) for 18 h. The gel is representative of three experiments with similar results.

    Article Snippet: In contrast, PARP1−/− macrophages exposed to LPS demonstrated no detectable HMGB1 levels in the supernatant ( ).

    Techniques: Activity Assay, Colorimetric Assay, Autoradiography, Western Blot, Concentration Assay

    RT-PCR of HMGB1 expression in THP-1 cells treated with LPS in the presence or absence of DIQ (300 μmol/L), a PARP1 inhibitor. THP-1 cells were treated with LPS (10 μg/mL) for 3 or 6 h. Error bars indicate mean ± SEM

    Journal: Molecular Medicine

    Article Title: Poly(ADP-Ribose) Polymerase 1–Sirtuin 1 Functional Interplay Regulates LPS-Mediated High Mobility Group Box 1 Secretion

    doi: 10.2119/molmed.2014.00156

    Figure Lengend Snippet: RT-PCR of HMGB1 expression in THP-1 cells treated with LPS in the presence or absence of DIQ (300 μmol/L), a PARP1 inhibitor. THP-1 cells were treated with LPS (10 μg/mL) for 3 or 6 h. Error bars indicate mean ± SEM

    Article Snippet: In contrast, PARP1−/− macrophages exposed to LPS demonstrated no detectable HMGB1 levels in the supernatant ( ).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

    (A) Representative confocal images of proximity ligation assay (PLA). Nuclear proteins were extracted from THP-1 cells treated with LPS in the presence or absence of DIQ (300 μmol/L), a PARP1 inhibitor. THP-1 cells were treated with LPS (10 μg/mL) for 6 h. PLA amplification corresponds with the interaction of PARP1 with SIRT1 and is visualized as red-pink spots localized mainly in the nucleus. (B) Coimmunoprecipitation analysis from THP-1 cell lysates. Panel 1: Samples were immunoprecipitated with anti-SIRT1 and immunoblotted with anti-poly(ADP-ribose). The blot was then stripped and reprobed for SIRT1 (panel 2).

    Journal: Molecular Medicine

    Article Title: Poly(ADP-Ribose) Polymerase 1–Sirtuin 1 Functional Interplay Regulates LPS-Mediated High Mobility Group Box 1 Secretion

    doi: 10.2119/molmed.2014.00156

    Figure Lengend Snippet: (A) Representative confocal images of proximity ligation assay (PLA). Nuclear proteins were extracted from THP-1 cells treated with LPS in the presence or absence of DIQ (300 μmol/L), a PARP1 inhibitor. THP-1 cells were treated with LPS (10 μg/mL) for 6 h. PLA amplification corresponds with the interaction of PARP1 with SIRT1 and is visualized as red-pink spots localized mainly in the nucleus. (B) Coimmunoprecipitation analysis from THP-1 cell lysates. Panel 1: Samples were immunoprecipitated with anti-SIRT1 and immunoblotted with anti-poly(ADP-ribose). The blot was then stripped and reprobed for SIRT1 (panel 2).

    Article Snippet: In contrast, PARP1−/− macrophages exposed to LPS demonstrated no detectable HMGB1 levels in the supernatant ( ).

    Techniques: Proximity Ligation Assay, Amplification, Immunoprecipitation

    Representative confocal images of THP-1 cells demonstrate the nuclear and cytoplasmic distribution of HMGB1 after LPS exposure in the absence (E–H) or presence of DIQ (300 μmol/L) (I–L). The cells were exposed to 10 μg/mL LPS in the absence or presence of 300 μmol/L DIQ. In these panels, the green staining indicates the presence of HMGB1 and the red staining indicates the presence of F-actin. The nuclear stain DRAQ5 is blue. Red arrows point to an increase in nuclear staining for HMGB1 with PARP1 inhibition.

    Journal: Molecular Medicine

    Article Title: Poly(ADP-Ribose) Polymerase 1–Sirtuin 1 Functional Interplay Regulates LPS-Mediated High Mobility Group Box 1 Secretion

    doi: 10.2119/molmed.2014.00156

    Figure Lengend Snippet: Representative confocal images of THP-1 cells demonstrate the nuclear and cytoplasmic distribution of HMGB1 after LPS exposure in the absence (E–H) or presence of DIQ (300 μmol/L) (I–L). The cells were exposed to 10 μg/mL LPS in the absence or presence of 300 μmol/L DIQ. In these panels, the green staining indicates the presence of HMGB1 and the red staining indicates the presence of F-actin. The nuclear stain DRAQ5 is blue. Red arrows point to an increase in nuclear staining for HMGB1 with PARP1 inhibition.

    Article Snippet: In contrast, PARP1−/− macrophages exposed to LPS demonstrated no detectable HMGB1 levels in the supernatant ( ).

    Techniques: Staining, Inhibition

    PARP1 inhibition abrogates LPS-mediated reduction in HDAC activity. Nuclear proteins were extracted from THP-1 cells treated with LPS in the presence or absence of DIQ (300 μmol/L), a PARP1 inhibitor. THP-1 cells were treated with LPS (10 μg/mL) for 4, 8 or 16 h. HDAC activity was determined by colorimetric assay. (*Represents P ≤ 0.05 versus LPS treated cells at the same time point.) Assay shown is representative of three experiments with similar results.

    Journal: Molecular Medicine

    Article Title: Poly(ADP-Ribose) Polymerase 1–Sirtuin 1 Functional Interplay Regulates LPS-Mediated High Mobility Group Box 1 Secretion

    doi: 10.2119/molmed.2014.00156

    Figure Lengend Snippet: PARP1 inhibition abrogates LPS-mediated reduction in HDAC activity. Nuclear proteins were extracted from THP-1 cells treated with LPS in the presence or absence of DIQ (300 μmol/L), a PARP1 inhibitor. THP-1 cells were treated with LPS (10 μg/mL) for 4, 8 or 16 h. HDAC activity was determined by colorimetric assay. (*Represents P ≤ 0.05 versus LPS treated cells at the same time point.) Assay shown is representative of three experiments with similar results.

    Article Snippet: In contrast, PARP1−/− macrophages exposed to LPS demonstrated no detectable HMGB1 levels in the supernatant ( ).

    Techniques: Inhibition, Activity Assay, Colorimetric Assay

    (A,B) Representative autoradiograph of Western blot analysis for supernatant HMGB1 levels in THP-1 cells treated with LPS in the presence or absence of DIQ (300 μmol/L), a PARP1 inhibitor (Figure 1A). Experiment was repeated by treating THP-1 cells with EB-47, a new potent PARP1 inhibitor and supernatant HMGB1 concentrations were analyzed (Figure 1B). THP-1 cells were treated with LPS (10 μg/mL) for 18 h. The gel is representative of three experiments with similar results. (C) Representative autoradiograph of Western blot analysis for supernatant HMGB1 levels in bone marrow–derived macrophages from WT and PARP1 −/− treated with LPS. The gel is representative of three experiments with similar results. (D) Representative Western blot analysis for supernatant HMGB1 concentrations in naïve, nontarget siRNA and PARP1 siRNA transfected THP-1 cells treated with LPS. Cells were treated with LPS (10 μg/mL) for 18 h. The gel is representative of three experiments with similar results. (E) PJ-34 (10 mg/kg, IP, n = 10), or saline (IP, n = 10) was administered 3 h prior to CLP. CLP was performed and blood was collected 24 h later. Serum HMGB1 concentrations were measured by ELISA.

    Journal: Molecular Medicine

    Article Title: Poly(ADP-Ribose) Polymerase 1–Sirtuin 1 Functional Interplay Regulates LPS-Mediated High Mobility Group Box 1 Secretion

    doi: 10.2119/molmed.2014.00156

    Figure Lengend Snippet: (A,B) Representative autoradiograph of Western blot analysis for supernatant HMGB1 levels in THP-1 cells treated with LPS in the presence or absence of DIQ (300 μmol/L), a PARP1 inhibitor (Figure 1A). Experiment was repeated by treating THP-1 cells with EB-47, a new potent PARP1 inhibitor and supernatant HMGB1 concentrations were analyzed (Figure 1B). THP-1 cells were treated with LPS (10 μg/mL) for 18 h. The gel is representative of three experiments with similar results. (C) Representative autoradiograph of Western blot analysis for supernatant HMGB1 levels in bone marrow–derived macrophages from WT and PARP1 −/− treated with LPS. The gel is representative of three experiments with similar results. (D) Representative Western blot analysis for supernatant HMGB1 concentrations in naïve, nontarget siRNA and PARP1 siRNA transfected THP-1 cells treated with LPS. Cells were treated with LPS (10 μg/mL) for 18 h. The gel is representative of three experiments with similar results. (E) PJ-34 (10 mg/kg, IP, n = 10), or saline (IP, n = 10) was administered 3 h prior to CLP. CLP was performed and blood was collected 24 h later. Serum HMGB1 concentrations were measured by ELISA.

    Article Snippet: In contrast, PARP1−/− macrophages exposed to LPS demonstrated no detectable HMGB1 levels in the supernatant ( ).

    Techniques: Autoradiography, Western Blot, Derivative Assay, Transfection, Enzyme-linked Immunosorbent Assay

    HCT116 PARP1 -/- cells are radiosensitive. (A) HCT116 PARP1 -/- cells (clones C2 and C4) and control PARP1-proficient HCT116 EV cells were exposed to IR (5 Gy) and counted after 48 hours. Bars represent the average and standard deviation of triplicates. Data is representative of two independent experiments. (B-C) Clonogenic assay after exposure to the indicated doses of IR. The surviving fraction is plotted in (B) and representative plates are shown in (C). Data is representative of two independent experiments. (D) Cell cycle profile after IR (5 Gy) at the indicated timepoints (hours after radiation). The percentage of cells with 2N and 4N DNA content is indicated. Data is representative of three independent experiments. (E-G) Quantification of irradiation-induced foci (IRIF). Cells were exposed to IR (2 Gy) and the number of foci per nucleus quantified by indirect immunofluorescence with antibodies to γ -H2AX and 53BP1. Histograms on (E) show the distribution of γ-H2AX foci per nucleus at baseline and 6 and 12 hours after IR. The percentage of cells with more than 10 γ-H2AX foci at the same timepoints is shown in F. Bars represent the average and standard deviation of three fields, N = 100 cells/field. (H) Cells extracts were harvested at the indicated timepoints after IR (4 Gy) and probed with antibodies to phospho-KAP1 (S824) and, as a loading control, GAPDH.

    Journal: PLoS ONE

    Article Title: PARP1 depletion induces RIG-I-dependent signaling in human cancer cells

    doi: 10.1371/journal.pone.0194611

    Figure Lengend Snippet: HCT116 PARP1 -/- cells are radiosensitive. (A) HCT116 PARP1 -/- cells (clones C2 and C4) and control PARP1-proficient HCT116 EV cells were exposed to IR (5 Gy) and counted after 48 hours. Bars represent the average and standard deviation of triplicates. Data is representative of two independent experiments. (B-C) Clonogenic assay after exposure to the indicated doses of IR. The surviving fraction is plotted in (B) and representative plates are shown in (C). Data is representative of two independent experiments. (D) Cell cycle profile after IR (5 Gy) at the indicated timepoints (hours after radiation). The percentage of cells with 2N and 4N DNA content is indicated. Data is representative of three independent experiments. (E-G) Quantification of irradiation-induced foci (IRIF). Cells were exposed to IR (2 Gy) and the number of foci per nucleus quantified by indirect immunofluorescence with antibodies to γ -H2AX and 53BP1. Histograms on (E) show the distribution of γ-H2AX foci per nucleus at baseline and 6 and 12 hours after IR. The percentage of cells with more than 10 γ-H2AX foci at the same timepoints is shown in F. Bars represent the average and standard deviation of three fields, N = 100 cells/field. (H) Cells extracts were harvested at the indicated timepoints after IR (4 Gy) and probed with antibodies to phospho-KAP1 (S824) and, as a loading control, GAPDH.

    Article Snippet: In contrast, PARP1 is dispensable for DSB repair per se via either Homologous Recombination (HR) or canonical NonHomologous End Joining (NHEJ).

    Techniques: Standard Deviation, Clonogenic Assay, Irradiation, Immunofluorescence

    HCT116 PARP1 -/- cells show defects in proliferation and increased senescence. (A) After editing PARP1 exon 2 via CRISPR/Cas9 D10A -dependent “double nicking”, single cell-derived subclones were screened by immunoblotting. Red asterisks mark PARP1 “knock out” (KO) clones. EV, extracts from HCT-116 cells transfected with an “empty vector” plasmid expressing Cas9 D10A but no gRNAs. Extracts from Parp1 -/- B cells were included as a negative control. (B) To quantify proliferation, 10 5 cells were seeded in 60 mm plates and counted at 24 and 48 hours. Data represents the mean and standard deviation of 3 independent experiments. (C-D) Clonogenic assay. Bars in (C) represent the average and standard deviation of 3 samples in 3 independent experiments. Representative examples are shown in (D). (E-F) Xenograft assay. HCT116 EV and HCT116 PARP1-/- cells (clone C4) were injected into NGS mice and tumors were measured at the indicated timepoints. Each data point in (E) represents the average and standard error of the mean (s.e.m.) of 10 tumors per line. Representative tumors at day 25 are shown in (F). (G-H) Cells were stained with senescence-associated β-galactosidase pH = 6.0 (SA β-gal) and the percentage of positive cells was quantified using brightfield microscopy. Data in (G) represents the average and standard deviation of 6 wells per cell line, N = 500 cells/well, pooled from two independent experiments. Representative examples are shown in (H). Black arrows point to senescent cells. As a positive technical control, HCT116 EV cells were treated with 10μM doxorubicin for 2 hours to induce senescence. (I-J) Cell cycle analysis after staining with propidium iodide (PI). Bars in (I) indicate the average and standard deviation of 3 independent experiments. Representative examples of cell cycle distributions are shown in (J). (K) Analyses of RNA-Seq data for members of the CDKN2 and CDKN1 families of cell cycle regulators. The fold change and p value were generated by comparing the expression of two KO clone to the EV control, as described in the Methods section. (L) Cell extracts were probed with antibodies to p21 and, as a loading control, GAPDH.

    Journal: PLoS ONE

    Article Title: PARP1 depletion induces RIG-I-dependent signaling in human cancer cells

    doi: 10.1371/journal.pone.0194611

    Figure Lengend Snippet: HCT116 PARP1 -/- cells show defects in proliferation and increased senescence. (A) After editing PARP1 exon 2 via CRISPR/Cas9 D10A -dependent “double nicking”, single cell-derived subclones were screened by immunoblotting. Red asterisks mark PARP1 “knock out” (KO) clones. EV, extracts from HCT-116 cells transfected with an “empty vector” plasmid expressing Cas9 D10A but no gRNAs. Extracts from Parp1 -/- B cells were included as a negative control. (B) To quantify proliferation, 10 5 cells were seeded in 60 mm plates and counted at 24 and 48 hours. Data represents the mean and standard deviation of 3 independent experiments. (C-D) Clonogenic assay. Bars in (C) represent the average and standard deviation of 3 samples in 3 independent experiments. Representative examples are shown in (D). (E-F) Xenograft assay. HCT116 EV and HCT116 PARP1-/- cells (clone C4) were injected into NGS mice and tumors were measured at the indicated timepoints. Each data point in (E) represents the average and standard error of the mean (s.e.m.) of 10 tumors per line. Representative tumors at day 25 are shown in (F). (G-H) Cells were stained with senescence-associated β-galactosidase pH = 6.0 (SA β-gal) and the percentage of positive cells was quantified using brightfield microscopy. Data in (G) represents the average and standard deviation of 6 wells per cell line, N = 500 cells/well, pooled from two independent experiments. Representative examples are shown in (H). Black arrows point to senescent cells. As a positive technical control, HCT116 EV cells were treated with 10μM doxorubicin for 2 hours to induce senescence. (I-J) Cell cycle analysis after staining with propidium iodide (PI). Bars in (I) indicate the average and standard deviation of 3 independent experiments. Representative examples of cell cycle distributions are shown in (J). (K) Analyses of RNA-Seq data for members of the CDKN2 and CDKN1 families of cell cycle regulators. The fold change and p value were generated by comparing the expression of two KO clone to the EV control, as described in the Methods section. (L) Cell extracts were probed with antibodies to p21 and, as a loading control, GAPDH.

    Article Snippet: In contrast, PARP1 is dispensable for DSB repair per se via either Homologous Recombination (HR) or canonical NonHomologous End Joining (NHEJ).

    Techniques: CRISPR, Derivative Assay, Transfection, Plasmid Preparation, Expressing, Negative Control, Standard Deviation, Clonogenic Assay, Xenograft Assay, Injection, Next-Generation Sequencing, Mouse Assay, Staining, Microscopy, Cell Cycle Assay, RNA Sequencing Assay, Generated

    PARP1 depletion and radiation synergistically induce ISGs and p21 expression. (A) HCT116 EV and HCT-116 PARP1-/- cells (clones C2 and C4) treated with IR (3 Gy) or control nonirradiated cells were harvested and mRNA for ISGs OAS1 and IFIT3 quantified by qRT-PCR. Bars represent the average and standard deviation of quadruplicates. Data is representative of two independent experiments. (B) Cells treated as in (A) and unirradiated controls were passaged for 15 days and OAS1 and IFIT3 mRNA were quantified by qRT-PCR in unirradiated cells (t = 0) and in irradiated cells at 1 day and 15 days after IR. Data was normalized to the expression of unirradiated HCT116 EV cells (= 1). (C) Cells were incubated with the JAK inhibitor ruxolitinib and OAS1 and IFIT3 mRNAs were quantified by qRT-PCR after radiation and in control mock-irradiated cells. (D) Extracts from cells treated as in (A) were probed with antibodies to p21, p53 or phospho-p53 (Ser15) at the indicated timepoints (hours after radiation). As a loading control, the same blots were probed with an antibody to β-actin. Lane 5, molecular weight marker.

    Journal: PLoS ONE

    Article Title: PARP1 depletion induces RIG-I-dependent signaling in human cancer cells

    doi: 10.1371/journal.pone.0194611

    Figure Lengend Snippet: PARP1 depletion and radiation synergistically induce ISGs and p21 expression. (A) HCT116 EV and HCT-116 PARP1-/- cells (clones C2 and C4) treated with IR (3 Gy) or control nonirradiated cells were harvested and mRNA for ISGs OAS1 and IFIT3 quantified by qRT-PCR. Bars represent the average and standard deviation of quadruplicates. Data is representative of two independent experiments. (B) Cells treated as in (A) and unirradiated controls were passaged for 15 days and OAS1 and IFIT3 mRNA were quantified by qRT-PCR in unirradiated cells (t = 0) and in irradiated cells at 1 day and 15 days after IR. Data was normalized to the expression of unirradiated HCT116 EV cells (= 1). (C) Cells were incubated with the JAK inhibitor ruxolitinib and OAS1 and IFIT3 mRNAs were quantified by qRT-PCR after radiation and in control mock-irradiated cells. (D) Extracts from cells treated as in (A) were probed with antibodies to p21, p53 or phospho-p53 (Ser15) at the indicated timepoints (hours after radiation). As a loading control, the same blots were probed with an antibody to β-actin. Lane 5, molecular weight marker.

    Article Snippet: In contrast, PARP1 is dispensable for DSB repair per se via either Homologous Recombination (HR) or canonical NonHomologous End Joining (NHEJ).

    Techniques: Expressing, Quantitative RT-PCR, Standard Deviation, Irradiation, Incubation, Molecular Weight, Marker

    ISG induction in cells surviving PARP1 depletion requires secreted factors and the α/β-IFN receptor. (A-B) HCT116 EV were incubated with conditioned media from either HCT116 EV or HCT116 PARP1 -/- cells (clones C2 and C4) for 48 hours and the number of cells counted in triplicates (A). In parallel, OAS1 and IFIT3 mRNAs were quantified by q-RT-PCR (B). Data is normalized to the expression in cells incubated with conditioned media from HCT116 EV cells. (C-D) HCT116 EV and HCT116 PARP1 -/- cells (clones C2 and C4) were incubated with an antibody to the α/β-IFN receptor or an isotype control for 96 hours and the number of cells counted in triplicates (C). For the same samples, mRNAs for β-IFN, several α-IFNs and the indicated ISGs were quantified by qRT-PCR (D). Data is normalized to expression in HCT116 EV cells treated with an isotype control. Data is representative of two independent experiments.

    Journal: PLoS ONE

    Article Title: PARP1 depletion induces RIG-I-dependent signaling in human cancer cells

    doi: 10.1371/journal.pone.0194611

    Figure Lengend Snippet: ISG induction in cells surviving PARP1 depletion requires secreted factors and the α/β-IFN receptor. (A-B) HCT116 EV were incubated with conditioned media from either HCT116 EV or HCT116 PARP1 -/- cells (clones C2 and C4) for 48 hours and the number of cells counted in triplicates (A). In parallel, OAS1 and IFIT3 mRNAs were quantified by q-RT-PCR (B). Data is normalized to the expression in cells incubated with conditioned media from HCT116 EV cells. (C-D) HCT116 EV and HCT116 PARP1 -/- cells (clones C2 and C4) were incubated with an antibody to the α/β-IFN receptor or an isotype control for 96 hours and the number of cells counted in triplicates (C). For the same samples, mRNAs for β-IFN, several α-IFNs and the indicated ISGs were quantified by qRT-PCR (D). Data is normalized to expression in HCT116 EV cells treated with an isotype control. Data is representative of two independent experiments.

    Article Snippet: In contrast, PARP1 is dispensable for DSB repair per se via either Homologous Recombination (HR) or canonical NonHomologous End Joining (NHEJ).

    Techniques: Incubation, Reverse Transcription Polymerase Chain Reaction, Expressing, Quantitative RT-PCR

    ISG induction in cells surviving PARP1 depletion is dependent on RIG-I and MAVS. (A-E) To assess the efficiency of RNA silencing to STING (A), RIG-I (B), MDA-5 (C), TLR3 (D) or MAVS (E), cells transfected with the specific pooled siRNAs and control cells transfected with a scrambled siRNA or untreated were harvested 4 days after transfection and probed with the indicated antibodies. GAPDH (A, B, C, E) or α-tubulin (D) served as loading controls. In blots where multiple bands are observed (A, D, E), black arrows point to specific bands and asterisks to unspecific bands. (F) The expression of ISGs OAS1 and IFIT3 was quantified by qRT-PCR after knock down. Data is normalized to the expression in HCT116 EV cells (= 1). Bars represent the average and standard deviation of quadruplicates. Data is representative of two independent experiments.

    Journal: PLoS ONE

    Article Title: PARP1 depletion induces RIG-I-dependent signaling in human cancer cells

    doi: 10.1371/journal.pone.0194611

    Figure Lengend Snippet: ISG induction in cells surviving PARP1 depletion is dependent on RIG-I and MAVS. (A-E) To assess the efficiency of RNA silencing to STING (A), RIG-I (B), MDA-5 (C), TLR3 (D) or MAVS (E), cells transfected with the specific pooled siRNAs and control cells transfected with a scrambled siRNA or untreated were harvested 4 days after transfection and probed with the indicated antibodies. GAPDH (A, B, C, E) or α-tubulin (D) served as loading controls. In blots where multiple bands are observed (A, D, E), black arrows point to specific bands and asterisks to unspecific bands. (F) The expression of ISGs OAS1 and IFIT3 was quantified by qRT-PCR after knock down. Data is normalized to the expression in HCT116 EV cells (= 1). Bars represent the average and standard deviation of quadruplicates. Data is representative of two independent experiments.

    Article Snippet: In contrast, PARP1 is dispensable for DSB repair per se via either Homologous Recombination (HR) or canonical NonHomologous End Joining (NHEJ).

    Techniques: Multiple Displacement Amplification, Transfection, Expressing, Quantitative RT-PCR, Standard Deviation

    HCT116 cells surviving PARP1 depletion activate innate immune signaling. (A-B) RNA-Seq data from HCT116 EV and HCT116 PARP1 -/- cells (clones C2 and C4) was analyzed using Gene Set Enrichment Analysis (GSEA). The top category of differentially expressed genes was “Interferon Alpha Response”. The enrichment plot is shown in (A) and the heat map for the 97 mRNAs in this category in shown in (B). Map was generated using Cufflinks software (version 2.2.1) and shows absolute expression values independently normalized and analyzed for each comparison pair (EV/C2 and EV/C4). (C) The same RNA-Seq dataset was analyzed using Ingenuity Pathway Analysis (IPA). A representative plot highlighting enrichment for Interferon-Stimulated Genes (ISGs) is shown. (D) The induction of multiple ISGs observed by RNA-Seq was confirmed by q-RT-PCR. Bars represent the average and standard deviation of quadruplicate samples in each experiment and data is representative of 2–4 independent experiments. (E-F) The induction of factors involved in the sensing/signaling of cytoplasmic nucleic acids observed by RNA-Seq was confirmed by Q-RT-PCR (E). Bars represent the average and standard deviation of quadruplicate samples in each experiment and data is representative of 2–3 independent experiments. Protein expression for the same factors was assessed by immunoblotting (F). Blots are representative of 2–3 independent experiments. (G) Fixed cells were stained with an antibody to IRF3 and counterstained with DAPI. Images are representative of 5 random fields per slide. The experiment was repeated twice with similar results.

    Journal: PLoS ONE

    Article Title: PARP1 depletion induces RIG-I-dependent signaling in human cancer cells

    doi: 10.1371/journal.pone.0194611

    Figure Lengend Snippet: HCT116 cells surviving PARP1 depletion activate innate immune signaling. (A-B) RNA-Seq data from HCT116 EV and HCT116 PARP1 -/- cells (clones C2 and C4) was analyzed using Gene Set Enrichment Analysis (GSEA). The top category of differentially expressed genes was “Interferon Alpha Response”. The enrichment plot is shown in (A) and the heat map for the 97 mRNAs in this category in shown in (B). Map was generated using Cufflinks software (version 2.2.1) and shows absolute expression values independently normalized and analyzed for each comparison pair (EV/C2 and EV/C4). (C) The same RNA-Seq dataset was analyzed using Ingenuity Pathway Analysis (IPA). A representative plot highlighting enrichment for Interferon-Stimulated Genes (ISGs) is shown. (D) The induction of multiple ISGs observed by RNA-Seq was confirmed by q-RT-PCR. Bars represent the average and standard deviation of quadruplicate samples in each experiment and data is representative of 2–4 independent experiments. (E-F) The induction of factors involved in the sensing/signaling of cytoplasmic nucleic acids observed by RNA-Seq was confirmed by Q-RT-PCR (E). Bars represent the average and standard deviation of quadruplicate samples in each experiment and data is representative of 2–3 independent experiments. Protein expression for the same factors was assessed by immunoblotting (F). Blots are representative of 2–3 independent experiments. (G) Fixed cells were stained with an antibody to IRF3 and counterstained with DAPI. Images are representative of 5 random fields per slide. The experiment was repeated twice with similar results.

    Article Snippet: In contrast, PARP1 is dispensable for DSB repair per se via either Homologous Recombination (HR) or canonical NonHomologous End Joining (NHEJ).

    Techniques: RNA Sequencing Assay, Generated, Software, Expressing, Indirect Immunoperoxidase Assay, Reverse Transcription Polymerase Chain Reaction, Standard Deviation, Staining