Journal: Nature Communications

Article Title: SPINDOC binds PARP1 to facilitate PARylation

doi: 10.1038/s41467-021-26588-y

Figure Lengend Snippet: a, b Hela cells were cotransfected with GFP-SPINDOC and RFP-PARP1 nanobody plasmids, 24 h later, cells were nontreated ( a ), or treated with PJ34 10 µM an hour ( b ), before being subjected to 355 nm UVA laser microirradiation-coupled live-cell imaging. Cells were treated with 200 ng/ml Hoechst 30 mins for presensitization prior to laser damage and imaging. Confocal images (40X) were taken every 5 s for 10 mins and representative images of the irradiation-path signals are shown. c Quantification of the corecruitment of GFP-SPINDOC and RFP-αPARP1 in a and b . Graphs represent mean ± SD, n = 10 biologically independent cells. d Hela-WT cells, Hela-SPINDOC KO cells, and the same cells rescued with GFP-SPINDOC and GFP-SPINDOC∆115–165aa mutant were transfected with RFP-αPARP1 nanobody plasmid and incubated for 24 h, before presensitizing with Hoechst (200 ng/ml), 355-nm UVA laser-induced damage and confocal imaging (40X). Images were taken every 20 s for 15 mins. e Quantification of RFP- αPARP1 recruitment in d . All timepoints are normalized to the peak of maximal recruitment of RFP-PARP1 to a value of 1.0 by each group. Graphs represent mean ± SD, n = 10 biologically independent cells. One-tailed Student t- tests were performed at 400 s, comparing Hela-WT with KO, P = 0.0118; Hela-KO vs KO-SPINDOC rescue, P = 0.0480; and Hela-KO vs KO - SPINDOC mutant, P = 0.0838. f Hela-SPINDOC WT and KO cell lines were treated with 10 Gy IR, allowed to recover for 0.5, 3, and 6 h, and subjected to Western blot analysis using the indicated antibodies. The data are representative of three independent experiments. g An in vitro PARylation assay was performed in the presence of purified GST, GST-SPINDOC or GST-SPINDOC115–165 proteins, NAD + , and recombinant PARP1, at room temperature for 30 min. Auto-PARylation signal was detected with αPAR antibody. The data are representative of three independent experiments. h PARP1 transcription level was evaluated by RT-qPCR in SPINDOC KO and overexpressed cells. One-tailed Student t -tests were performed, SPINDOC KO vs WT, P = 0.1822; Flag-SPINDOC vs vector, P = 0.2270. i Hela-SPINDOC WT and KO cell lines were treated with 10-Gy IR, allowed to recover for 0.5, 3, and 6 h, and subjected to RT-qPCR assay for the six indicated targets. Graphs represent mean ± SD, n = 3 biologically independent samples. One-tailed Student t -tests were performed, SPINDOC WT, ** P = 0.0044, * P = 0.0121, *** P < 0.001, **** P < 0.0001. SPINDOC KO, RAD51: * P = 0.0382, ** P = 0.0091. NRF1: * P = 0.1027, 0.0871, 0.0302. NF-κB1: P = 0.0018, 0.0093, 0.3797; P 130: P = 0.0304, 0.0054, 0.0066; hNIS: P = 0.0154, 0.0068, 0.0030. j Hela-SPINDOC KO cells rescued with GFP-SPINDOC, pEGFP-C1 as a control, were treated with 10-Gy IR, allowed to recover for 0.5, 3, and 6 h, and subjected to RT-qPCR assay. Graphs represent mean ± SD, n = 3 biologically independent samples. One-tailed Student t -tests were performed, SPINDOC KO vector, BRCA1, P = 0.0.0046, 0.0043; NRF1: ** P = 0.0025; P130: ** P = 0.0095; hNIS: * P = 0.0321. SPINDOC KO-GFP-S P INDOC, RAD51: ** P = 0.0018; NRF1: ** P = 0.0038; NF-κB1: ** P = 0.0042; P130: ** P = 0.0013, 0.0080; *** P < 0.001, **** P < 0.0001, NS: nonsignificant.

Article Snippet: Media was changed 4~6 h after transfected cells and cells were cultured for another 24 h. Next, cells were treated with (or without) PJ34 (Selleckchem, Cat# S7300) 10μM an hour and then changed the media with 200 ng/mL Hoechst33342 (Thermo Scientific, Cat# 62249) for 30 mins prior to laser-stripe microirradiation and imaging.

Techniques: Live Cell Imaging, Imaging, Irradiation, Mutagenesis, Transfection, Plasmid Preparation, Incubation, One-tailed Test, Western Blot, In Vitro, Purification, Recombinant, Quantitative RT-PCR, Control

Journal: bioRxiv

Article Title: Parthanatos-inducing zinc agent C010DS-Zn elicits anti-tumor immune responses involving T cells and macrophages in vivo

doi: 10.1101/2021.03.18.433812

Figure Lengend Snippet: Basic substance information on C010DS-Zn, and its control substances C005D-Zn, zinc sulfate (ZnSO 4 ), and “Free Zn-Pyr (7:1)” (the mixture of zinc sulfate and sodium pyrithione at zinc-to-pyrithione molar ratio of 7-to-1). (A) Molecular structure of C010DS, 34 kDa (M w ) γPGA polymer each conjugated with 1 Cy5.5 label, 2 PEG-folate sidechains, 2 PEG-cRGDfK sidechains, and 10 PEG-S-S-pyrithione sidechains. C010DS-Zn preparation is described in the Methods. (B) Molecular structure of C005D, 34 kDa (M w ) γPGA polymer each conjugated with 1 Cy5.5 label, 3 PEG-folate sidechains, and 3 PEG-cRGDfK sidechains. C005D-Zn preparation is described in the Methods. (C) Comparative in vitro cytotoxicity evaluation on the tested compounds using LDH release assays after 24h treatments against 4T1 cells. (D) Results of the in vitro time-resolved apoptosis-necrosis flow cytometry assay at fixed doses against 4T1 cells using A5 and PI labels. (E) Results of the in vitro dose-resolved apoptosis-necrosis flow cytometry assay at 3h time point against 4T1 cells using A5 and PI labels. (F) In vitro PAR-ELISA assay results on the C010DS-Zn treated 4T1 cells with or without the PARP inhibitor PJ34. * p <0.05. ¶ Indicated group displayed significantly higher PAR signal than the Control, 2.5 μM Zn, or 5 μM Zn C010DS-Zn treatment groups at p <0.05 with or without PJ34 co-treatment.

Article Snippet: PJ34 (Santacruz Biotech Cat# sc-204161A) stock solution was prepared at 2mM concentration in water.

Techniques: Control, Polymer, In Vitro, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Parthanatos-inducing zinc agent C010DS-Zn elicits anti-tumor immune responses involving T cells and macrophages in vivo

doi: 10.1101/2021.03.18.433812

Figure Lengend Snippet: In vitro attenuation of 1.5h C010DS-Zn treatment effects against 4T1 including nuclear AIF translocation, DNA fragmentation, and cell viability loss by PARP inhibitor PJ34. (A) Representative confocal fluorescence images of the 4T1 cells treated for 1.5h with vehicle, ZnSO4, C005D-Zn, or C010DS-Zn at different concentrations, labelled with Hoechst (blue: adherent cell count), TUNEL (green: DNA breaks), and anti-AIF (red), with or without the PARP inhibitor PJ34 co-incubation. Bar=100μm. (B) Quantitative analysis of the fluorescence images for cell viability, nuclear AIF translocation, and nuclear TUNEL intensity using CirAvgInten function. 3 imaging fields quantified for each group (n=3). * p <0.05. ** p <0.005. *** p <0.0005. ¶Indicated group’s average value is significantly greater than those of all other treatment groups minimally at p <0.05. C010DS-Zn at 60 μM Zn treatment group resulted in complete cell loss, and hence its quantitative imaging analyses could not be performed.

Article Snippet: PJ34 (Santacruz Biotech Cat# sc-204161A) stock solution was prepared at 2mM concentration in water.

Techniques: In Vitro, Translocation Assay, Fluorescence, Cell Counting, TUNEL Assay, Incubation, Imaging

Journal: Antioxidants

Article Title: Neurotoxic Effect of Myricitrin in Copper-Induced Oxidative Stress Is Mediated by Increased Intracellular Ca 2+ Levels and ROS/p53/p38 Axis

doi: 10.3390/antiox14010046

Figure Lengend Snippet: Effects of specific inhibitors and modulators of intracellular signalling pathways on the neurotoxic effect of myricitrin. Cells were treated simultaneously with 0.5 mM CuSO 4 and 20 µg/mL myricitrin in combination with the following inhibitors: pifithrin-α (p53 inhibitor) ( A ), PJ34 (PARP inhibitor) ( B ), wortmannin (PI3K/Akt inhibitor) ( C ), U0126 (ERK1/2 inhibitor) ( D ), SB203580 (p38 inhibitor) ( E ), SP600125 (JNK inhibitor) ( F ), BAPTA-AM (intracellular calcium chelator) ( G ), MK-801 (NMDA receptor channel blocker) ( H ), nifedipine L-type calcium channel inhibitor) ( I ), and leupeptin (calpain inhibitor) ( J ). Inhibitors were applied 1 h prior to and during the 24 h treatment with copper and myricitrin. Data are presented as means ± SD from 3 to 5 independent experiments performed in triplicate. * p < 0.05, ** p < 0.01, and **** p < 0.0001 vs. copper + myricitrin treatment (one-way ANOVA followed by post hoc Tukey’s test).

Article Snippet: MK-801 hydrogen maleate, pifithrin-α, SP600125, SB203580, and PARP inhibitor VIII (PJ34) were purchased from Alfa Aesar (Ward Hill, MA, USA).

Techniques:

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: 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