Journal: Cell reports

Article Title: Protein folding stress potentiates NLRP1 and CARD8 inflammasome activation

doi: 10.1016/j.celrep.2022.111965

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: PARP rabbit polyclonal Ab , Cell Signaling Tech , Cat# 9542; RRID: AB_2160739.

Techniques: Recombinant, LDH Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay, Viability Assay, Cytotoxicity Assay, Software

Journal: Cell reports

Article Title: Protein folding stress potentiates NLRP1 and CARD8 inflammasome activation

doi: 10.1016/j.celrep.2022.111965

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: PARP rabbit polyclonal Ab , Cell Signaling Tech , Cat# 9542; RRID: AB_2160739.

Techniques: Recombinant, LDH Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay, Viability Assay, Cytotoxicity Assay, Software

Journal: Molecular Therapy Oncolytics

Article Title: Upregulation of endogenous TRAIL-elicited apoptosis is essential for metformin-mediated antitumor activity against TNBC and NSCLC

doi: 10.1016/j.omto.2021.04.012

Figure Lengend Snippet: Metformin induced apoptosis in both TNBC and NSCLC cells (A) TNBC cells (HCC70, MDA-MB-468, and BT549) and NSCLC cells (H460, H1650, and A549) were plated onto 6 cm dishes with culture medium containing 10% FBS. After 24 h, cells were treated with indicated concentrations of metformin in fresh medium with 5% FBS for 48 h. Both adherent and non-adherent cells were collected and subjected to apoptosis analysis using a cell death detection ELISA. Data show a representative of three independent experiments. Bars, SD. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (B) The same batch of cell lysates was used for western blot analyses with specific antibodies directed against PARP, caspase-8, caspase-3, or β-actin.

Article Snippet: Antibodies for western blot analyses were from the following sources: PARP rabbit mAb (46D11), caspase-8 mouse mAb (1C12), caspase-3 rabbit mAb (8G10), DR4 rabbit mAb (D9S1R), DR5 rabbit mAb (D4E9) (Cell Signaling Technology, Beverly, MA, USA); TRAIL mouse mAb (B35-1) (R&D Systems); and β-actin mouse mAb (clone AC-75) (Sigma Chemical, St. Louis, MO, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Molecular Therapy Oncolytics

Article Title: Upregulation of endogenous TRAIL-elicited apoptosis is essential for metformin-mediated antitumor activity against TNBC and NSCLC

doi: 10.1016/j.omto.2021.04.012

Figure Lengend Snippet: Inhibition of TRAIL function with a recombinant TRAIL-R2 Fc chimera significantly attenuated metformin-induced apoptosis in TNBC and NSCLC cells (A) TNBC and NSCLC cells were plated onto 6 cm dishes with medium containing 10% FBS. After 24 h, the culture medium was replaced with fresh medium with 5% FBS containing either metformin or TRAIL-R2 Fc chimera protein alone or combinations of metformin and TRAIL-R2 Fc chimera for 48 h. Both adherent and non-adherent cells were collected and subjected to apoptosis ELISA. Data show a representative of three independent experiments. Bars, SD. ns, not significant, ∗p < 0.05, ∗∗p < 0.01. (B) The same batch of cell lysates was used for western blot analyses with specific antibodies directed against PARP, caspase-8, caspase-3, or β-actin.

Article Snippet: Antibodies for western blot analyses were from the following sources: PARP rabbit mAb (46D11), caspase-8 mouse mAb (1C12), caspase-3 rabbit mAb (8G10), DR4 rabbit mAb (D9S1R), DR5 rabbit mAb (D4E9) (Cell Signaling Technology, Beverly, MA, USA); TRAIL mouse mAb (B35-1) (R&D Systems); and β-actin mouse mAb (clone AC-75) (Sigma Chemical, St. Louis, MO, USA).

Techniques: Inhibition, Recombinant, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Molecular Therapy Oncolytics

Article Title: Upregulation of endogenous TRAIL-elicited apoptosis is essential for metformin-mediated antitumor activity against TNBC and NSCLC

doi: 10.1016/j.omto.2021.04.012

Figure Lengend Snippet: Specific knockdown of TRAIL expression inhibited metformin-induced apoptosis in TNBC and NSCLC cells TNBC and NSCLC cells were infected with lentivirus containing either control shRNA (sh-scr) or specific TRAIL -targeting shRNA (sh-1 or sh-2). The infected cells were selected by puromycin for 24 h and then treated with or without metformin for additional 48 h. (A) Both adherent and non-adherent cells were collected and subjected to western blot analyses of TRAIL, PARP, caspase-8, caspase-3, or β-actin. The densitometry analyses of TRAIL signals were shown underneath, and the arbitrary numbers indicated the intensities of each cell line relative to controls, defined as 1.0. (B) The same batch of cell lysates was subjected to apoptosis ELISA. Data show a representative of three independent experiments. Bars, SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.01, ∗∗∗∗p < 0.001.

Article Snippet: Antibodies for western blot analyses were from the following sources: PARP rabbit mAb (46D11), caspase-8 mouse mAb (1C12), caspase-3 rabbit mAb (8G10), DR4 rabbit mAb (D9S1R), DR5 rabbit mAb (D4E9) (Cell Signaling Technology, Beverly, MA, USA); TRAIL mouse mAb (B35-1) (R&D Systems); and β-actin mouse mAb (clone AC-75) (Sigma Chemical, St. Louis, MO, USA).

Techniques: Knockdown, Expressing, Infection, Control, shRNA, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy Oncolytics

Article Title: Upregulation of endogenous TRAIL-elicited apoptosis is essential for metformin-mediated antitumor activity against TNBC and NSCLC

doi: 10.1016/j.omto.2021.04.012

Figure Lengend Snippet: Metformin inhibited tumor growth and induced apoptosis and TRAIL expression in a tumor xenograft model (A) Tumor growth curves were plotted using average tumor volume within each group at the indicated time points. A two-tailed Student’s t test was used for statistical analysis (∗p < 0.01, ∗∗p < 0.003). Bars: SD. (B and C) At the end of treatment, tumor-bearing mice from control group and metformin-treated group were sacrificed. The tumors were dissected, imaged as indicated (B), and measured for weight (C). (D and E) Cell lysis from the tumor tissues was prepared. Western blot assays were performed to examine the expression of TRAIL, DR4, DR5, and apoptotic markers PARP, caspase-8, and caspase-3. β-actin was used as an internal control. The densitometry analyses of TRAIL signals are shown underneath, and the arbitrary numbers indicate the intensities of each tumor relative to control 1, defined as 1.0. (F) Formalin-fixed paraffin-embedded sections of xenograft tumors were analyzed with H&E staining, IHC staining for Ki67, TRAIL, and cleaved caspase-3. Scale bar, 210 μm. Quantification of IHC staining with ImageJ and ImageJ plugin IHC profiler are shown underneath.

Article Snippet: Antibodies for western blot analyses were from the following sources: PARP rabbit mAb (46D11), caspase-8 mouse mAb (1C12), caspase-3 rabbit mAb (8G10), DR4 rabbit mAb (D9S1R), DR5 rabbit mAb (D4E9) (Cell Signaling Technology, Beverly, MA, USA); TRAIL mouse mAb (B35-1) (R&D Systems); and β-actin mouse mAb (clone AC-75) (Sigma Chemical, St. Louis, MO, USA).

Techniques: Expressing, Two Tailed Test, Control, Lysis, Western Blot, Formalin-fixed Paraffin-Embedded, Staining, Immunohistochemistry

Journal: Cell Reports Medicine

Article Title: Single-cell analysis of neoplastic plasma cells identifies myeloma pathobiology mediators and potential targets

doi: 10.1016/j.xcrm.2024.101925

Figure Lengend Snippet:

Article Snippet: PARP (46D11) Rabbit mAb , Cell Signaling Technology , 9532; RRID: AB_659884.

Techniques: Recombinant, Selection, Cell Isolation, Proliferation Assay, Staining, Enzyme-linked Immunosorbent Assay, Detection Assay, Reporter Assay, RNA Sequencing, Transgenic Assay, Software

Journal: The Journal of Cell Biology

Article Title: MEIS homeodomain proteins facilitate PARP1/ARTD1-mediated eviction of histone H1

doi: 10.1083/jcb.201701154

Figure Lengend Snippet: MEIS and PARP1 interact. (A) Mass spectrometry scores of PARP1 and PARP1-interacting proteins copurifying with MEIS2 from nuclear extracts of SK-N-BE(2) neuroblastoma cells. (B and C) PARP1 (B) and MEIS2 (C) protein distribution in the RMS. MEIS2 and PARP1 protein staining is shown in brown, and nuclear counterstaining is in blue. CC, corpus callosum. (D) Domain structure of PARP1. (E) GST pulldown of HA-tagged MEIS2 with different PARP1–GST fusion proteins. The left panel shows a blot probed for HA detecting MEIS2-HA, and the right panel shows the same blot probed for GST detecting the different PARP1–GST fusion proteins. Because transfer of full-length PARP1–GST was incomplete because of its large size, higher-exposure images of the blot probed for HA and GST are shown in Fig. S2. The number of biological replicates performed for E are listed in Table S4. PD, pulldown; WB, Western blot.

Article Snippet: Human recombinant PARP1 enzyme (Enzo Life Sciences) was incubated with biotinylated NAD + (Trevigen) in 50 mM Tris, pH 8, 25 mM MgCl 2 , and 1 mM DTT in the presence or absence of 2.5 µg sonicated linear double-stranded salmon sperm DNA (Sigma Aldrich) per reaction as indicated.

Techniques: Mass Spectrometry, Staining, Western Blot

Journal: The Journal of Cell Biology

Article Title: MEIS homeodomain proteins facilitate PARP1/ARTD1-mediated eviction of histone H1

doi: 10.1083/jcb.201701154

Figure Lengend Snippet: MEIS recruits PARP1 to the Dcx promoter/enhancer. (A) ChIP-qPCR for PARP1 at Dcx(−2.7) during neuronal differentiation. (B) Comparison of PARP1 binding to Dcx(−2.7) and “ primers out ” (−12) at 0 and 5 h; values for Dcx(−2.7) correspond with those shown in A. (C) ChIP-qPCR for H1 at Dcx(−2.7) and “ primers out ” at the times indicated. (D) ChIP-qPCR for PARP1 and H1 at the Myog promoter, showing reciprocal binding of PARP1 and H1. (E and F) ChIP-reChIP for H1 followed by PAR at Dcx(−2.7) (E) and “ primers out ” (F) at 3 h of differentiation. (G) In vitro PARylation assay of recombinant PARP1 and H1 in the presence of biotinylated (biot.) NAD + and stimulated by the addition of low-molecular DNA fragments (sheared DNA), demonstrating efficient PARylation of H1 and autoPARylation of PARP1. The asterisks in A–F indicate statistically significant enrichment of ChIP with the antibodies indicated relative to ChIP with the IgG control antibodies for the same conditions, with *, P < 0.05; **, P < 0.01; ***, P < 0.001. Statistical significance of ChIP results between experimental groups is given as p = numerical value. ChIP data are represented as means ± SEM. Samples sizes and the number of biological replicates are listed in Table S4. IP, immunoprecipitation; WB, Western blot.

Article Snippet: Human recombinant PARP1 enzyme (Enzo Life Sciences) was incubated with biotinylated NAD + (Trevigen) in 50 mM Tris, pH 8, 25 mM MgCl 2 , and 1 mM DTT in the presence or absence of 2.5 µg sonicated linear double-stranded salmon sperm DNA (Sigma Aldrich) per reaction as indicated.

Techniques: Binding Assay, In Vitro, Recombinant, Immunoprecipitation, Western Blot

Journal: The Journal of Cell Biology

Article Title: MEIS homeodomain proteins facilitate PARP1/ARTD1-mediated eviction of histone H1

doi: 10.1083/jcb.201701154

Figure Lengend Snippet: MEIS recruits PARP1 to Dcx(−2.7) . (A and B) ChIP-qPCR for the proteins indicated at 5 h of differentiation in cells transfected with Meis1/2 -specific siRNAs (gray bars) or nontargeting control siRNAs (black bars): MEIS, PARP1, and PBX1 at Dcx(−2.7) (A), and H1 at Dcx(−2.7) (B) . (C) ChIP-qPCR for H1 at Dcx(−2.7) in undifferentiated cell cultures. H1 is not released from the Dcx promoter/enhancer when cells are subjected to the cellular differentiation protocol under Meis -knockdown conditions. The asterisks indicate statistically significant enrichment of ChIP with the antibodies indicated relative to ChIP with the IgG control antibodies for the same conditions, with *, P < 0.05; **, P < 0.01; ***, P < 0.001. Statistical significance of ChIP results between experimental groups is given as p = numerical value. ChIP data are represented as means ± SEM. Samples sizes and the number of biological replicates are listed in Table S4.

Article Snippet: Human recombinant PARP1 enzyme (Enzo Life Sciences) was incubated with biotinylated NAD + (Trevigen) in 50 mM Tris, pH 8, 25 mM MgCl 2 , and 1 mM DTT in the presence or absence of 2.5 µg sonicated linear double-stranded salmon sperm DNA (Sigma Aldrich) per reaction as indicated.

Techniques: Transfection, Cell Differentiation

Journal: The Journal of Cell Biology

Article Title: MEIS homeodomain proteins facilitate PARP1/ARTD1-mediated eviction of histone H1

doi: 10.1083/jcb.201701154

Figure Lengend Snippet: PARP activity is required for neuronal differentiation and H1 eviction from the Dcx promoter/enhancer. (A) Schematic outline of the experiments shown in B–F. (B–F) Reduced neurogenesis and enhanced astrogliogenesis upon pharmacological PARP inhibition: (B and C) Proportions of neurons (B) and astrocytes (C) generated in the presence of increasing concentrations of Olaparib. (D) Representative images of cultures differentiated in the presence of Olaparib or 0.01% DMSO as control. Arrowheads indicate DCX-positive neuronal processes. (E and F) Neurons and astrocytes generated in the presence of 3AB (E) or PJ34 (F). (G, left) Outline of the experiment; (right) neuronal differentiation after shRNA-mediated knockdown of PARP1. (H, left) Outline of the experiment; (right) qPCR for Meis2 , Pbx1 , and Dcx transcripts in cells differentiated for 10 h by growth factor removal and plating on laminin in the presence of 100 nM Olaparib. Expression is shown relative to expression determined in cells treated with 0.01% DMSO (vehicle only). (I, left) Outline of the experiment; (right) ChIP-qPCR for H1 at Dcx(−2.7) in cells differentiated for 5 h in the presence of 6 mM 3AB (gray bars) or water as vehicle control (black bars). Statistical significance of ChIP results between experimental groups is given as p = numerical value. Data are represented as means ± SEM. Samples sizes and the number of biological replicates are listed in Table S4.

Article Snippet: Human recombinant PARP1 enzyme (Enzo Life Sciences) was incubated with biotinylated NAD + (Trevigen) in 50 mM Tris, pH 8, 25 mM MgCl 2 , and 1 mM DTT in the presence or absence of 2.5 µg sonicated linear double-stranded salmon sperm DNA (Sigma Aldrich) per reaction as indicated.

Techniques: Activity Assay, Inhibition, Generated, shRNA, Expressing

Journal: The Journal of Cell Biology

Article Title: MEIS homeodomain proteins facilitate PARP1/ARTD1-mediated eviction of histone H1

doi: 10.1083/jcb.201701154

Figure Lengend Snippet: Neuronal differentiation is not compromised when pharmacological inhibition of PARP follows the induction of cellular differentiation. (A, left) Outline of the experiment: differentiation was induced in primary SVZ progenitor cells by growth factor removal and plating on laminin 12 h before addition of PJ34 to the culture medium. Addition of water (diluent) served as control; (right) proportion of TuJ1 + neurons generated under both conditions; after 3 d of differentiation, 53.9 ± 3.6% (SD) of the cells differentiated into DCX + neurons under standard conditions, and 49.9 ± 7.6% (SD) differentiated when PJ34 was added to the medium after the first 12 h of differentiation. (B) Representative micrographs of these experiments. Data are represented as means ± SEM, and the number of biological replicates is listed in Table S4.

Article Snippet: Human recombinant PARP1 enzyme (Enzo Life Sciences) was incubated with biotinylated NAD + (Trevigen) in 50 mM Tris, pH 8, 25 mM MgCl 2 , and 1 mM DTT in the presence or absence of 2.5 µg sonicated linear double-stranded salmon sperm DNA (Sigma Aldrich) per reaction as indicated.

Techniques: Inhibition, Cell Differentiation, Generated

Journal: The Journal of Cell Biology

Article Title: MEIS homeodomain proteins facilitate PARP1/ARTD1-mediated eviction of histone H1

doi: 10.1083/jcb.201701154

Figure Lengend Snippet: Identification of PARP-regulated genes by Affymetrix Mouse Gene 1.0 ST arrays. (A) Scatter blot of differentially regulated genes in adult SVZ progenitor cells differentiated in the presence or absence of 100 nM Olaparib; representative significantly up-regulated genes are highlighted in red, and representative significantly down-regulated genes are in green. A list of all significantly differentially expressed genes is given in Table S5. (B) GO term enrichment analysis for genes differentially expressed after Olaparib treatment relative to the control. A more detailed collection of GO terms is shown in Table S1. (C) Transcript expression of six down-regulated candidate genes in SVZ-derived stem and progenitor cells differentiated for 10 h in vitro upon pharmacological inhibition of PARP1 and PARP2 or Meis1/2 knockdown, respectively, validated by qPCR. Gray bars, PARP-inhibition: transfection with nontargeting siRNAs and treatment with 100 nM Olaparib; black bars, Meis knockdown: transfection with Meis1/2 -specific siRNAs and treatment with 0.01% DMSO. Transcript levels are expressed relative to those determined under control conditions (transfection with nontargeting siRNAs and differentiation in the presence of 0.01% DMSO). (D) ChIP-qPCR for MEIS2 and PARP1 at a consensus-binding motif for MEIS/PBX-type HD proteins upstream of the Draxin transcriptional start site in adult SVZ progenitor cells after 5 h of neuronally directed differentiation. Gray bars, cells transfected with Meis1/2 -specific siRNAs; black bars, cells transfected with nontargeting control siRNAs. (E) ChIP-qPCR for MEIS2 and PARP1 under identical conditions as shown in D but binding to a consensus motif for MEIS/PBX-type HD proteins upstream of the Nrep transcriptional start site was assessed. (F–I) Transcript expression for Draxin (F and G) and Nrep (H and I) in the adult SVZ visualized by in situ hybridization in comparison with MEIS2 and DCX protein expression. (F and H) MEIS2 (red) and DCX (green) cell nuclei are counterstained with DAPI (blue). (G and H) Overlay of transcript expression (purple) and MEIS2 protein (red). The number of biological replicates is listed in Table S4.

Article Snippet: Human recombinant PARP1 enzyme (Enzo Life Sciences) was incubated with biotinylated NAD + (Trevigen) in 50 mM Tris, pH 8, 25 mM MgCl 2 , and 1 mM DTT in the presence or absence of 2.5 µg sonicated linear double-stranded salmon sperm DNA (Sigma Aldrich) per reaction as indicated.

Techniques: Expressing, Derivative Assay, In Vitro, Inhibition, Transfection, Binding Assay, In Situ Hybridization

Journal: Cell Reports Medicine

Article Title: Cholinergic signaling via muscarinic M1 receptor confers resistance to docetaxel in prostate cancer

doi: 10.1016/j.xcrm.2023.101388

Figure Lengend Snippet: CHRM1 regulates DTX efficacy in PC cells (A) Cell viability assays of control and CHRM1-OE 22Rv1 and DU145 cells upon DTX treatment for 48 h (n = 4 biological replicates), with CHRM1 OE validated by western blots. (B) Cell viability assay of 22Rv1 cells expressing hM1D q , a CHRM1-specific DREADD-Gα q construct, under pre-treatment with CNO (20 μM, 24 h) followed by DTX for 24 h (n = 4 biological replicates). (C) Western blot of cleaved PARP1 and caspase 3 in control and CHRM1-OE 22Rv1 and DU145 cells treated with DTX (1 nM, 48 h). (D) Western blot of cleaved PARP1 and caspase 3 in 22Rv1 cells treated with or without McN-A-343 (50 μM, 24 h) followed by DTX (1 nM, 48 h). (E) Cell viability assays of 22Rv1 and DU145 cells pre-treated with or without Dic (25 μM, 24 h) followed by DTX for 48 h (n = 4 biological replicates). (F) Soft-agar colony-formation assays of 22Rv1 and DU145 cells treated with 1 nM DTX together with or without 25 μM Dic for 48 h followed by a 2-week cell growth period (n = 3 biological replicates). (G) Western blot of cleaved PARP1 and caspase 3 in 22Rv1 cells treated with or without Dic (10 or 25 μM, 24 h) followed by DTX (1 nM, 48 h). (H) TUNEL assays of 22Rv1 and DU145 cells treated with or without Dic (25 μM, 24 h) followed by DTX (1 nM, 48 h) (n = 3 biological replicates). Scale bars, 200 μm. Data represent mean ± SD. ∗∗p < 0.01.

Article Snippet: Rabbit polyclonal anti-cleaved PARP1 , Cell Signaling Technology , Cat# 9541; RRID: AB_331426.

Techniques: Control, Western Blot, Viability Assay, Expressing, Construct, TUNEL Assay

Journal: Cell Reports Medicine

Article Title: Cholinergic signaling via muscarinic M1 receptor confers resistance to docetaxel in prostate cancer

doi: 10.1016/j.xcrm.2023.101388

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-cleaved PARP1 , Cell Signaling Technology , Cat# 9541; RRID: AB_331426.

Techniques: Western Blot, Double Staining Immunohistochemistry, Staining, Virus, shRNA, Control, Subcloning, Recombinant, Reverse Transcription, Transfection, Bicinchoninic Acid Protein Assay, Cell Viability Assay, In Situ, Calcium Flux Assay, Enzyme-linked Immunosorbent Assay, Gel Extraction, Mutagenesis, Purification, Ligation, Software

Journal: Oncogene

Article Title: Interaction of hepatitis B virus X protein with PARP1 results in inhibition of DNA repair in hepatocellular carcinoma.

doi: 10.1038/onc.2016.82

Figure Lengend Snippet: Figure 1. HBx interacts with PARP1 through its C-terminus. (a) Chang cells were transfected with the expression vector for FLAG-HBx and immunoprecipitation was performed using anti-FLAG antibody. Immunoprecipitated proteins including PARP1 (arrow) were visualized by Coomassie staining after sodium dodecyl sulfate–polyacrylamide gel electrophoresis. (b) Cell lysates obtained from SNU354 and SNU398 cells that expressing HBx endogenously were immunoprecipitated (IP) with anti-HBx or anti-PARP1 and probed by western blotting (WB) with anti-PARP1 or anti-HBx as indicated. PHEN, an inhibitor of PARP1, was employed as a control. (c) Expression of HBx and PARP1 in SNU354 cells were visualized by immunofluorescence staining. DAPI was used to stain the nuclei (magnification, × 400). Yellow bars represent 10 μm. (d) A schematic representation of PARP1 domains (top). Chang cells were transfected with the expression vectors for Myc-HBx and FLAG-tagged full- length (Full), DNA-binding domain (DBD), BRCA1 C-terminal domain (BRCT), or catalytic domain (CatD) of PARP1, or empty vector (EV). Cell lysates were immunoprecipitated (IP) with anti-FLAG, and probed by WB with anti-Myc antibody (bottom). (e) A schematic representation of HBx domains (top). Chang cells were transfected with the expression vectors for HA-PARP1 and Myc-tagged full-length (Full), N-terminus (NT), or C-terminus (CT) of HBx, or EV. Cell lysates were immunoprecipitated with anti-Myc and probed by WB with anti-HA antibody (bottom).

Article Snippet: Cell lysates were obtained at various times after transfection, and the ChIP assay was performed using specific antibodies against Myc, PARP1 (Santa Cruz), p95 NBS1 (#3002; Cell Signaling Technology), Sirt6, or γH2AX (Abcam).

Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Staining, Polyacrylamide Gel Electrophoresis, Western Blot, Control, Binding Assay

Journal: Oncogene

Article Title: Interaction of hepatitis B virus X protein with PARP1 results in inhibition of DNA repair in hepatocellular carcinoma.

doi: 10.1038/onc.2016.82

Figure Lengend Snippet: Figure 2. HBx inhibits PARP1 enzymatic activity in vivo and in vitro. (a) Chang X-34 cells treated with 4 μg/ml doxycycline (Doxy) for 24 h, or Chang, Huh-7 or HepG2 cells transfected with the expression vector for Myc-HBx. And then cells were treated with 0.5 mM H2O2 for 5 min. Expression of PAR and the indicated proteins was analyzed by western blotting. (b) In vitro PARylation assay was performed using the purified recombinant GST-PARP1 (220 ng) and GST-HBx-His (rhHBx) proteins (0.5, 1 and 3 μg) with NAD+. The PARylated PARP1 was detected by western blotting using anti-PAR antibody. Expression levels of GST-PARP1 and GST-HBx-His were shown as control. (c) Chang cells transfected with empty vector (EV) or expression vector for full-length (Full), N-terminus (NT) or C-terminus (CT) of HBx. PARP activities in the cell lysates were analyzed by a colorimetric ELISA. *Po0.05 compared with empty vector (EV) (n = 3).

Article Snippet: Cell lysates were obtained at various times after transfection, and the ChIP assay was performed using specific antibodies against Myc, PARP1 (Santa Cruz), p95 NBS1 (#3002; Cell Signaling Technology), Sirt6, or γH2AX (Abcam).

Techniques: Activity Assay, In Vivo, In Vitro, Transfection, Expressing, Plasmid Preparation, Western Blot, Recombinant, Control, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: Interaction of hepatitis B virus X protein with PARP1 results in inhibition of DNA repair in hepatocellular carcinoma.

doi: 10.1038/onc.2016.82

Figure Lengend Snippet: Figure 3. HBx also interacts with Sirt6 and thereby interrupts the interaction between Sirt6 and PARP1. Chang cells were seeded in 150 mm culture dishes. (a) The cells were transfected with FLAG-Sirt6, HA-PARP1 and Myc-HBx. Cell lysates were immunoprecipitated (IP) with anti-HA or anti-Myc, and probed by western blotting (WB) with the indicated antibodies. (b) Chang X-34 cells were treated with 4 μg/ml doxycycline. The interaction of Sirt6 and PARP1 (red) was visualized by PLA. DAPI was used to stain the nuclei. The mean numbers of dots (mean ± s.e.m.) were 15.65 ± 0.52 and 7.52 ± 0.33 in the vehicle (Veh)-treated and doxycycline (Doxy)-treated groups, respectively. ***Po0.001 compared as indicated (n = 3). The expression of indicated proteins in the cell lysates was probed by WB as control. (c) A schematic representation of Sirt6 domains and deleted constructs (top). Chang cells were transfected with the expression vectors for HA-HBx and Myc-tagged full-length (Full), N-terminal deletion (ΔN), C-terminal deletion (ΔC), catalytic core (Core) of Sirt6 or empty vector (EV). Cell lysates were immunoprecipitated (IP) with anti-Myc, and probed by WB with anti-HA antibody. (d) Chang cells were transfected with the expression vectors for Myc-Sirt6 and HA- tagged full-length (Full), N-terminus (NT), or C-terminus (CT) of HBx, or empty vector (EV). (e) Chang cells were transfected with 3 μg FLAG- tagged PARP1-BRCT, 3 μg Myc-tagged Sirt6-N and together with the increasing amounts of HA-tagged HBx-CT. Cell lysates were obtained and immunoprecipitated with anti-FLAG or anti-HA, and probed by WB with the indicated antibodies. (f) A model for the HBx-induced interruption of PARP1 binding to Sirt6.

Article Snippet: Cell lysates were obtained at various times after transfection, and the ChIP assay was performed using specific antibodies against Myc, PARP1 (Santa Cruz), p95 NBS1 (#3002; Cell Signaling Technology), Sirt6, or γH2AX (Abcam).

Techniques: Transfection, Immunoprecipitation, Western Blot, Staining, Expressing, Control, Construct, Plasmid Preparation, Binding Assay

Journal: Oncogene

Article Title: Interaction of hepatitis B virus X protein with PARP1 results in inhibition of DNA repair in hepatocellular carcinoma.

doi: 10.1038/onc.2016.82

Figure Lengend Snippet: Figure 4. HBx inhibits DNA repair of the paraquat-induced DNA damage. Chang cells were seeded in 24-well plate and transfected with 0.5 μg each the expression vector for full-length (Full), N-terminus (NT) or C-terminus (CT) of HBx and treated with 1 mM paraquat (PQ) for 16 h. The cells were harvested at the indicated recovery time. (a) The γH2AX foci were visualized by immunofluor- escence staining at the indicated recovery time (RT). DAPI was used to stain the nuclei. (b, c) The number of γH2AX foci in nucleus of at least 100 cells was counted at the indicated recovery time. The values are expressed as the mean ± s.d. ***Po0.001 compared with empty vector (EV) at each time (n = 3). (d) Chang cells were transfected with the indicated combination of expression vectors for HBx, PARP1 and Sirt6 for 24 h. The cells were harvested at 24 h of recovery time. *Po0.05, **Po0.01, and ***Po0.001 compared with empty vector (EV). The values are expressed as the mean ± s.d. ###Po0.001 compared with full-length HBx with empty vector (EV). ¶¶¶Po0.001 compared to CT-HBx with empty vector (EV) (n = 3).

Article Snippet: Cell lysates were obtained at various times after transfection, and the ChIP assay was performed using specific antibodies against Myc, PARP1 (Santa Cruz), p95 NBS1 (#3002; Cell Signaling Technology), Sirt6, or γH2AX (Abcam).

Techniques: Transfection, Expressing, Plasmid Preparation, Staining

Journal: Oncogene

Article Title: Interaction of hepatitis B virus X protein with PARP1 results in inhibition of DNA repair in hepatocellular carcinoma.

doi: 10.1038/onc.2016.82

Figure Lengend Snippet: Figure 6. HBx augments the ROS-induced oxidative DNA damage. (a) Chang X-34 cells that pretreated with 4 μg/ml doxycycline (Doxy) for 24 h were treated with 0.5 mM H2O2 for 5 min. The amount of 8-OHdG (8-hydroxy-2′-deoxyguanosine) in cell lysates was quantified by ELISA. The values are expressed as the mean ± s.d. *Po0.05 compared with vehicle treatment; #Po0.05 compared as indicated (n = 3). (b) Chang cells that transfected with Myc-HBx were treated with 1 mM paraquat (PQ) for 24 h with or without continued treatment of 0.5 mM H2O2 for 5 min. Double-stranded DNA damage was detected by the neutral comet assays. Representative images of comet formation are shown (left). Tail moment of comet formations per 100–150 cells was estimated (right). The values are expressed as the mean ± s.d. ***Po0.001 for no treatment; ###Po0.001 compared as indicated. (c) SNU398 cells were transfected with siRNA-HBx for 48 h and then the cells were treated with 0.5 mM H2O2 for 5 min. Tail moment of comet formations per 100–150 cells was scored. The values are expressed as the mean ± s.d. ***Po0.001 compared with without H2O2; ###Po0.001 compared with vehicle or H2O2 treatment with siGL3, respectively. (d) Chang cells were transfected with the indicated combination of expression vectors for HBx, PARP1 and Sirt6 for 24 h and then the cells were treated with 1 mM paraquat for 24 h. Tail moment of comet formations per 100–150 cells was scored. The values are expressed as the mean ± s.d. **Po0.01 and ***Po0.001 compared with without HBx in each group. ###Po0.001 compared with HBx with empty vector (EV). ¶¶¶Po0.001 compared with HBx, paraquat and empty vector (EV) (n = 3).

Article Snippet: Cell lysates were obtained at various times after transfection, and the ChIP assay was performed using specific antibodies against Myc, PARP1 (Santa Cruz), p95 NBS1 (#3002; Cell Signaling Technology), Sirt6, or γH2AX (Abcam).

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Expressing, Plasmid Preparation

Journal: eLife

Article Title: Functional identification of soluble uric acid as an endogenous inhibitor of CD38

doi: 10.7554/eLife.96962

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Article Snippet: Commercial assay or kit , HT Universal Colorimetric PARP Assay Kit , R&D Systems , Cat#4677-096-K , .

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Staining, Colorimetric Assay, Software, Activity Assay