Journal: Frontiers in Molecular Neuroscience

Article Title: Alteration in Intracellular Zn 2+ Homeostasis as a Result of TRPM2 Channel Activation Contributes to ROS-Induced Hippocampal Neuronal Death

doi: 10.3389/fnmol.2017.00414

Figure Lengend Snippet: TRPM2 channel is crucial for H 2 O 2 -induced production of mitochondrial reactive oxygen species (ROS) in hippocampal neurons. (A,C) Representative fluorescence images showing MitoTracker Red CM-H 2 Xros (MitoROS) fluorescence in WT (left column) or TRPM2-KO (right column) neurons under control (CTL) and treatment with 100 μM or 300 μM H 2 O 2 for 2 h (A) , or in WT neurons treated with 1 μM PJ34 or 1 μM TPEN, 30 min before and during exposure to 300 μM H 2 O 2 (C) . Scale bar is 100 μm. (B,D) Mean MitoROS fluorescence intensity from 3 to 5 independent experiments with 35–70 neurons examined in each experiment, presented relative to the fluorescence level in control neurons in matched experiments. 2-APB was used at 100 μM. * p < 0.05 and *** p < 0.005 indicate significant difference from control. ††† p < 0.005 indicates significant difference from neurons exposed to 300 μM H 2 O 2 alone.

Article Snippet: PJ34 was from Calbiochem, and TPEN from StressMarq Biosciences.

Techniques: Fluorescence

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: To inhibit PARP1 enzymatic activity during the differentiation process, the medium was supplemented with PJ34 hydrochloride (Tocris Bioscience), 3AB (Sigma-Aldrich), or Olaparib (AZD2281; Lynparza; Selleckchem) in the listed concentrations.

Techniques: Activity Assay, Inhibition, Generated, Control, shRNA, Knockdown, Expressing, ChIP-qPCR

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: To inhibit PARP1 enzymatic activity during the differentiation process, the medium was supplemented with PJ34 hydrochloride (Tocris Bioscience), 3AB (Sigma-Aldrich), or Olaparib (AZD2281; Lynparza; Selleckchem) in the listed concentrations.

Techniques: Inhibition, Cell Differentiation, Control, Generated

Journal: Nucleic Acids Research

Article Title: Nuclear poly(A)-binding protein and nucleolin utilize their RNA recognition motifs to read PAR chains

doi: 10.1093/nar/gkaf1090

Figure Lengend Snippet: PARylation-dependent recruitment of NCL and PABPN1 to DNA damage sites. ( A ) Live-cell imaging snapshots showing ectopic GFP-NCL and GFP-PABPN1 recruitment to laser-induced DNA damage sites in U2OS cells. Recruitment was blocked by PARP1 inhibition with PJ34. Endogenous PARP1 was detected using an RFP-fused nanobody. ( B ) Binding of NCL to PARP1 in U2OS cells was promoted by H 2 O 2 and H 2 O 2 /PARGi treatments. DNase and RNase treatment did not impact the NCL/PARP1 interaction. An anti-Flag IP was performed on lysates from Flag-PARP1 overexpressed cells, followed by immunoblotting with Flag and NCL antibodies. ( C ) Binding of PABPN1 to PARP1 in U2OS cells was promoted by H 2 O 2 and H 2 O 2 /PARGi treatment. DNase and RNase treatment did not impact the PABPN1/PARP1 interaction. An anti-Flag IP was performed on lysates from Flag-PARP1 and GFP-PABPN1 co-expressed cells, followed by immunoblotting with Flag and GFP antibodies.

Article Snippet: Stock solutions of etoposide (Sigma, Cat #E1383), PARGi (PDD 00017273, MedChemExpress, Cat #HY-108360) and PJ34 (Selleckchem, Cat #S7300) were prepared in DMSO (Dimethyl sulfoxide).

Techniques: Live Cell Imaging, Inhibition, Binding Assay, Western Blot

Journal: Nucleic Acids Research

Article Title: Nuclear poly(A)-binding protein and nucleolin utilize their RNA recognition motifs to read PAR chains

doi: 10.1093/nar/gkaf1090

Figure Lengend Snippet: The RRM of PABPN1 binds PAR and RNA competitively. ( A, B ) Pull-down analysis shows that the mutation of two aromatic residues (mR) in the RRM of PABPN1 blocks poly(A) RNA binding ( A ) but not PAR-long chain binding ( B ). ( C ) Live-cell imaging snapshots show the recruitment of ectopic GFP-PABPN1 and mutant GFP-PABPN1-mR to laser-induced DNA damage sites in U2OS cells. Recruitment was blocked by the PARP1 inhibitor PJ34. ( D ) Kinetic characterization of RNA and PAR binding to the RRMs of PABPN1 and NCL utilizing single cycle SPR. ( E ) FP assays showed that PABPN1 RRM binding to PAR and RNA is competitive in vitro . GST-RRM of PABPN1 was either incubated with poly(A) and titrated with increasing concentrations of PAR5 (PAR5 competition; black trace) or incubated with PAR5 and titrated with increasing concentrations of poly(A) (RNA competition; blue trace). ( F, G ) Pull-down assays demonstrate that PABPN1 RRM binding to PAR and RNA is competitive in vitro . GST-RRM of PABPN1 was either incubated with biotinylated PAR-long overnight, followed by incubation with varying concentrations of poly(A) for 2 h ( F ), or incubated with biotinylated poly(A) overnight, followed by incubation with varying concentrations of PAR-long for 2 h ( G ). Samples were analyzed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and western blotting with an anti-GST antibody. Poly(A) 8-mer RNA was used in all experiments.

Article Snippet: Stock solutions of etoposide (Sigma, Cat #E1383), PARGi (PDD 00017273, MedChemExpress, Cat #HY-108360) and PJ34 (Selleckchem, Cat #S7300) were prepared in DMSO (Dimethyl sulfoxide).

Techniques: Mutagenesis, RNA Binding Assay, Binding Assay, Live Cell Imaging, In Vitro, Incubation, Polyacrylamide Gel Electrophoresis, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Transcriptional Reprogramming and Resistance to Colonic Mucosal Injury in Poly(ADP-ribose) Polymerase 1 (PARP1)-deficient Mice *

doi: 10.1074/jbc.M116.714386

Figure Lengend Snippet: Loss of protective effects of Parp1 deficiency in Parp1−/−x Rag2−/− DKO mice. A, body weight loss and mortality in response to treatment with 4% DSS for 7 days. B, representative H&E histology staining of the proximal and distal colon of Rag2−/− and DKO mice treated with H2O or 3% DSS for 7 days. C, mucosal TNFα and IFNγ expression in the proximal and distal colon of Rag2−/− and DKO mice treated with 3% DSS for 7 days (N.S., not statistically significant).

Article Snippet: Experimental Animals Specific pathogen-free wild-type (WT) 129/SvEv mice, Parp1 −/− and Rag2 −/− mice on the same genetic background, were originally obtained from Taconic (Germantown, NY).

Techniques: Staining, Expressing