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  • 95
    Millipore anti parp
    Anti Parp, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti parp
    (A) Resveratrol, rapamycin and a combination of the two inhibit mTORC1 and <t>mTORC2</t> signaling in the MM1.S cell line, as determined by immunoblot analysis. (B) Immunoblot analysis for caspase-3, <t>PARP,</t> p-Rb and cyclin D1 in MM1.S cells treated with resveratrol, rapamycin or a combination of the two. mTORC, mammalian transcriptional coactivator for CREB; PARP, poly (ADP-ribose) polymerase; p-Rb, phosphorylated retinoblastoma protein; Akt, protein kinase B.
    Anti Parp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 4471 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc parp
    BCR-ABL deregulates miR-126 biogenesis (a,b) pri-miR-126 (n=8 biologically independent samples) (a) and pre-miR-126 (n=8 biologically independent samples) (b) expression levels, as assessed by QPCR, in the indicated human normal and CML cell populations. (c) BCR-ABL and SPRED1 staining in CML CD34 + cells by immunofluorescence (IF). (d) Immunoprecipitation (IP) with anti-SPRED1 followed by immunoblotting (IB) with anti-SPRED1 and anti-phosphotyrosine (p-Tyr) antibodies (left) and an in vitro kinase assay (right), as performed by IP with anti-c-Abl or anti-normal mouse IgG as control and immunoblotting with anti-SPRED1, in lysates of K562 cells treated with none, DMSO (vehicle) or NIL. (e) IP with <t>anti-RAN</t> followed by IB with anti-SPRED1 and anti-RAN antibodies in lysates of K562 cells. (f) SPRED1 and RAN staining by IF in K562 cells treated with none, DMSO or NIL. (g) SPRED1, RAN, RCC1 and Exp-5 expression in cytoplasmic (Cyt) and nuclear (Nu) fractions from K562 cells, treated with DMSO or NIL, as assessed by IB. Densitometric quantification of selected bands is shown (normalized to the actin loading control for total and Cyt lysates or to the <t>PARP</t> loading control for Nu lysates). (h) IP with anti-RAN followed by IB with anti-SPRED1, RAN, Exp-5 and RCC1 antibodies in lysates of K562 cells, CML CD34 + cells, and normal CD34+ cells treated with DMSO or NIL. Densitometric quantification of selected bands is shown (normalized to the actin loading control). (i) Mature, pri- and pre-miR-126 expression, as assessed by QPCR, in K562 and CML CD34 + cells treated with DMSO or NIL (n=3 independent experiments for K562 and 3 independent samples for CML cells). (j) IP with anti-RAN followed by IB with anti-SPRED1, Exp-5, RCC1 and RAN antibodies in lysates of K562 cells without or with washing-off of NIL. Densitometric quantification of selected bands is shown (normalized to the actin loading control). (k–m) Mature and pri-miR-126 expression as assessed by QPCR (n=3 independent experiments) (k), miR-126 staining (l), and mature and pre-miR-126 levels as assessed by Northern blotting (m) in K562 cells with or without washing-off of NIL. (n) IP with anti-RAN followed by IB with anti-Exp-5, RCC1 and RAN antibodies in lysates of K562 and CML CD34 + . Comparison between groups was performed by two-tailed, unpaired Student’s t -test. P values ≤0.05 were considered significant. Results shown represent mean ± SEM. * p ≤ 0.05, ** p
    Parp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 11643 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/parp/product/Cell Signaling Technology Inc
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    93
    Santa Cruz Biotechnology anti parp
    JNK2, but not JNK1, is essential for virus-induced apoptosis. ( A ) Control, JNK1 and JNK2 siRNA knock-down HEK293 cells ( left ), or wild type, Jnk1 −/− and Jnk2 −/− MEF cells ( right ), were treated with SeV (for HEK293 MOI = 1, for MEF MOI = 4) for the indicated times. Cell lysates were analyzed by western blot, probing for ISG15, ISG60, JNK1 and JNK2 with the indicated antibodies. ( B ) Wild type and Mavs −/− MEF cells were treated with SeV (MOI = 4), or TNF-α (10 ng/ml) plus cycloheximide (CHX, 10 µg/ml) for the indicated times. Cell lysates were collected for western blot analysis using <t>anti-PARP</t> antibody to determine cell apoptosis and using anti-MAVS antibody to measure the deficiency of MAVS protein. ( C ) HEK293 cells were transfected with the indicated plasmids and 24 hours later, cell lysates were collected for western blot analysis of PARP, phosphorylated JNK, phosphorylated IRF3, Flag-tagged proteins and β-actin. ( D ) HEK293 cells were treated by SeV (MOI = 1) with or without JNK kinase inhibitor SP600125 (5 µM). Cell lysates were collected for western blot analysis of PARP, cleaved <t>caspase-3</t> and β-actin to probe for cell apoptosis. ( E ) Control, JNK1 or JNK2 knocked down HEK293 cells were treated with SeV (MOI = 1) for the indicated times. Cell lysates were collected for western blot analysis to measure cell apoptosis using the indicated antibodies. ( F ) Wild type, Jnk1 −/− or Jnk2 −/− MEF cells were treated with SeV (MOI = 4) for the indicated times. Cell lysates were collected for western blot analysis.
    Anti Parp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 848 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson anti parp
    Targeting of <t>RAD51</t> activates several signaling pathways but attenuated by combining with <t>PARP</t> and p38 inhibition A. The Human Phospho-Kinase arrays (R D Systems) were probed with MDA-MB-231 lysate samples that had been treated for 72 hours; samples used are labeled (from top to bottom): (1) DMSO treatment, (2) 10 μM RAD51i, (3) 2.5 μM PARPi/10 μM p38i, and (4) 10 μM RAD51i /2.5 μM PARPi/10 μM p38i triple combination. Highlighted dots represent a significant change in signal over DMSO treated controls. The corners are positive control blots for quantification. B. Quantitation of spot intensity was standardized for cells treated with DMSO and plotted as normalized intensity. Several kinases displayed greater than 2 fold increase in phosphorylation compared to references. Shading represents grouping based on pathway signaling. C. Protein expression and changes in phosphorylation of ERK1/2, p38, STAT3, MK-2 (p38 target) and AKT were confirmed by western blotting.
    Anti Parp, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 412 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology anti parp 1
    3-AB inhibits mouse MOR mRNA expression in NS20Y cells and schematic model for <t>PARP-1</t> in modulation of mouse MOR transcription. (A) Quantification of transcripts was performed by RT-PCR. Total RNA from NS20Y cells treated with 2 mM 3-AB was prepared and treated with DNase I. Primer pairs specific for the coding sequence of each gene were used for RT-PCR. PCR products were visualized in a 2% agarose gel. Lane 1: Molecular weight markers (M); lane 2: Control; lane 3: 3-AB-treated cells. (B) Quantitative analysis using ImageQuant 5.2 software. The MOR mRNA levels from Control and 3-AB-treated cells were normalized against β-actin levels. The values were obtained from triplicate data points. Changes in transcript levels for 3-AB-treated samples were compared to Control, which was assigned a value of 1.0. Bars indicate the range of standard error. (C) Schematic model for the role of PARP-1 in modulation of mouse MOR gene transcription. In neuronal cells, enzymatically active PARP-1 interacts strongly with the poly(C) sequence of the mouse MOR promoter and aids in the formation of tran-scriptionally inactive chromatin. Enzymatic inhibition of PARP-1 by 3-AB results in non-poly(ADP-ribosyl)ated PARP-1 and subsequently, an increase in the levels of MOR mRNA in mouse NS20Y cells.
    Anti Parp 1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 593 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc rabbit polyclonal anti parp
    3-AB inhibits mouse MOR mRNA expression in NS20Y cells and schematic model for <t>PARP-1</t> in modulation of mouse MOR transcription. (A) Quantification of transcripts was performed by RT-PCR. Total RNA from NS20Y cells treated with 2 mM 3-AB was prepared and treated with DNase I. Primer pairs specific for the coding sequence of each gene were used for RT-PCR. PCR products were visualized in a 2% agarose gel. Lane 1: Molecular weight markers (M); lane 2: Control; lane 3: 3-AB-treated cells. (B) Quantitative analysis using ImageQuant 5.2 software. The MOR mRNA levels from Control and 3-AB-treated cells were normalized against β-actin levels. The values were obtained from triplicate data points. Changes in transcript levels for 3-AB-treated samples were compared to Control, which was assigned a value of 1.0. Bars indicate the range of standard error. (C) Schematic model for the role of PARP-1 in modulation of mouse MOR gene transcription. In neuronal cells, enzymatically active PARP-1 interacts strongly with the poly(C) sequence of the mouse MOR promoter and aids in the formation of tran-scriptionally inactive chromatin. Enzymatic inhibition of PARP-1 by 3-AB results in non-poly(ADP-ribosyl)ated PARP-1 and subsequently, an increase in the levels of MOR mRNA in mouse NS20Y cells.
    Rabbit Polyclonal Anti Parp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 309 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Abcam anti parp
    The function of miR-142-3p in sorafenib resistance is dependent on the regulation of <t>ATG5</t> and ATG16L1. a MTT assay was performed to measure the sensitivity of SMMC-7721 cells co-transfected with miR-142-3p mimics and ATG5 or ATG16L1 to sorafenib. b Colony-formation assay was employed to determine the proliferation ability of SMMC-7721 cells co-transfected with miR-142-3p mimics and ATG5 or ATG16L1. c Flow cytometric assay was employed to measure the apoptosis rate in SMMC-7721 cells co-transfected with miR-142-3p mimics and ATG5 or ATG16L1. d Western blotting was utilized to assess the levels of the apoptosis-related proteins c-caspase-3 and <t>c-PARP</t> and autophagy-related proteins in SMMC-7721 cells co-transfected with miR-142-3p mimics and ATG5 or ATG16L1. All data are presented as the mean ± S.D. from three independent experiments. The p -values represent comparisons between groups (** p
    Anti Parp, supplied by Abcam, used in various techniques. Bioz Stars score: 97/100, based on 234 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Promega anti parp p85 fragment pab
    The function of miR-142-3p in sorafenib resistance is dependent on the regulation of <t>ATG5</t> and ATG16L1. a MTT assay was performed to measure the sensitivity of SMMC-7721 cells co-transfected with miR-142-3p mimics and ATG5 or ATG16L1 to sorafenib. b Colony-formation assay was employed to determine the proliferation ability of SMMC-7721 cells co-transfected with miR-142-3p mimics and ATG5 or ATG16L1. c Flow cytometric assay was employed to measure the apoptosis rate in SMMC-7721 cells co-transfected with miR-142-3p mimics and ATG5 or ATG16L1. d Western blotting was utilized to assess the levels of the apoptosis-related proteins c-caspase-3 and <t>c-PARP</t> and autophagy-related proteins in SMMC-7721 cells co-transfected with miR-142-3p mimics and ATG5 or ATG16L1. All data are presented as the mean ± S.D. from three independent experiments. The p -values represent comparisons between groups (** p
    Anti Parp P85 Fragment Pab, supplied by Promega, used in various techniques. Bioz Stars score: 96/100, based on 300 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc poly adp ribose polymerase
    Induction of apoptosis by alkylaminoguaiazulenes in HSC-2 cells. Near-confluent HSC-2 cells were incubated for 24 h with the indicated concentrations of alkylaminoguaiazulenes and expression of cleaved products of poly <t>ADP-ribose</t> polymerase (PARP) and <t>caspase-3</t> was visualized by western blot analysis. Each sample contains 0.1% DMSO. Act.D., Actinomycin D (1 μM), was used as a positive control for apoptosis.
    Poly Adp Ribose Polymerase, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1441 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Becton Dickinson mouse anti parp
    ZEB1 promotes EMT . A-C . Immunoblots. PANC-1 cells were pre-treated with TGF-β1 for two days and then transfected twice (day 0 and day 2) with ZEB1 siRNAs in the continued presence of TGF-β1. Four days after the initial transfection, cells were harvested. A . By up-regulating epithelial proteins such as E-cadherin and CAR, knockdown of ZEB1 antagonizes <t>TGF-β-induced</t> EMT in PANC-1 cells. Similarly, silencing of ZEB1 expression in MDA-MB-231 cells up-regulates E-cadherin and CAR, and down-regulates the mesenchymal marker fibronectin. B . PANC-1 cells were treated with TGF-β1, and harvested at the indicated time-points for analysis of the total protein fractions. C . PANC-1 cells were treated with TGF-β1 and subjected to cell fractionation. Abbreviations: C, TGF-β1 solvent control [4 mM HCl/0.1% (v/w) BSA]; UT, untransfected; Ctrl #1, siControl ON-TARGETplus Non-targeting siRNA #1 (Dharmacon); Ctrl #2, firefly luciferase-targeting siRNA; ZEB1 siRNA #1/#2, ZEB1-targeting siRNAs. Ctrl #2 and ZEB1 siRNA sequences are provided in Additional file 1 (Table S3). Chinese Hamster Ovary cells stably expressing human CAR (CHO+), or vector (CHO-) [ 9 ]. Loading controls are shown as β-actin, β-tubulin, GAPDH and <t>PARP</t> signals, with GAPDH as a cytoplasmic, and PARP as a nuclear marker.
    Mouse Anti Parp, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 89/100, based on 85 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


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

    Journal: Oncology Letters

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

    doi: 10.3892/ol.2018.8178

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

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

    Techniques:

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

    Journal: Molecular Medicine

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

    doi: 10.2119/molmed.2014.00102

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

    Article Snippet: Samples were analyzed by Western blotting by using antibodies recognizing different parts of the proteins: the C terminus of RNF146 (Aviva Systems Biology, San Diego, CA, USA) and the region surrounding Gly215 of PARP-1 (Cell Signaling Technology) and protein-A-HRP conjugate (Amersham/GE Healthcare, Life-Sciences, Pittsburgh, PA, USA).

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

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

    Journal: Molecular Medicine

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

    doi: 10.2119/molmed.2014.00102

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

    Article Snippet: Samples were analyzed by Western blotting by using antibodies recognizing different parts of the proteins: the C terminus of RNF146 (Aviva Systems Biology, San Diego, CA, USA) and the region surrounding Gly215 of PARP-1 (Cell Signaling Technology) and protein-A-HRP conjugate (Amersham/GE Healthcare, Life-Sciences, Pittsburgh, PA, USA).

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

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

    Journal: Molecular Medicine

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

    doi: 10.2119/molmed.2014.00102

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

    Article Snippet: Samples were analyzed by Western blotting by using antibodies recognizing different parts of the proteins: the C terminus of RNF146 (Aviva Systems Biology, San Diego, CA, USA) and the region surrounding Gly215 of PARP-1 (Cell Signaling Technology) and protein-A-HRP conjugate (Amersham/GE Healthcare, Life-Sciences, Pittsburgh, PA, USA).

    Techniques: In Vivo, Western Blot, CTL Assay

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

    Journal: Molecular Medicine

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

    doi: 10.2119/molmed.2014.00102

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

    Article Snippet: Samples were analyzed by Western blotting by using antibodies recognizing different parts of the proteins: the C terminus of RNF146 (Aviva Systems Biology, San Diego, CA, USA) and the region surrounding Gly215 of PARP-1 (Cell Signaling Technology) and protein-A-HRP conjugate (Amersham/GE Healthcare, Life-Sciences, Pittsburgh, PA, USA).

    Techniques: Expressing, Labeling, Staining

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

    Journal: Molecular Medicine

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

    doi: 10.2119/molmed.2014.00102

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

    Article Snippet: Samples were analyzed by Western blotting by using antibodies recognizing different parts of the proteins: the C terminus of RNF146 (Aviva Systems Biology, San Diego, CA, USA) and the region surrounding Gly215 of PARP-1 (Cell Signaling Technology) and protein-A-HRP conjugate (Amersham/GE Healthcare, Life-Sciences, Pittsburgh, PA, USA).

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

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

    Journal: Molecular Medicine

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

    doi: 10.2119/molmed.2014.00102

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

    Article Snippet: Samples were analyzed by Western blotting by using antibodies recognizing different parts of the proteins: the C terminus of RNF146 (Aviva Systems Biology, San Diego, CA, USA) and the region surrounding Gly215 of PARP-1 (Cell Signaling Technology) and protein-A-HRP conjugate (Amersham/GE Healthcare, Life-Sciences, Pittsburgh, PA, USA).

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

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

    Journal: Molecular Medicine

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

    doi: 10.2119/molmed.2014.00102

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

    Article Snippet: Samples were analyzed by Western blotting by using antibodies recognizing different parts of the proteins: the C terminus of RNF146 (Aviva Systems Biology, San Diego, CA, USA) and the region surrounding Gly215 of PARP-1 (Cell Signaling Technology) and protein-A-HRP conjugate (Amersham/GE Healthcare, Life-Sciences, Pittsburgh, PA, USA).

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

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

    Journal: Molecular Medicine

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

    doi: 10.2119/molmed.2014.00102

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

    Article Snippet: Samples were analyzed by Western blotting by using antibodies recognizing different parts of the proteins: the C terminus of RNF146 (Aviva Systems Biology, San Diego, CA, USA) and the region surrounding Gly215 of PARP-1 (Cell Signaling Technology) and protein-A-HRP conjugate (Amersham/GE Healthcare, Life-Sciences, Pittsburgh, PA, USA).

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

    Effect of the combination of LDM and CQ on apoptosis pathways. H460 cells were treated with both CQ and LDM (CQ pretreatment for 2 h) or none for 20 h. zVAD.fmk (100 μmol/L) were added before CQ and LDM for 1.5 h. (A) zVAD.fmk partly reversed the inhibitory effect of CQ combined with LDM on the proliferation of H460 cells. Mean±SD. n =3. (B) zVAD.fmk reduced the increasing expression of cleaved-PARP and cleaved-caspase 3 induced by CQ and LDM. (C) H460 cells were treated with CQ (50 μmol/L), LDM (0.5 nmol/L) and both (CQ pretreatment for 2 h) for 20 h. The influence of LDM and CQ (alone or both) on expression of p53, Bax, and Bcl-2. The expression levels of Bax and Bcl-2 were measured as the density by the Gel-Pro software, standardized by the density of β-actin.

    Journal: Acta Pharmacologica Sinica

    Article Title: Chloroquine potentiates the anti-cancer effect of lidamycin on non-small cell lung cancer cells in vitro

    doi: 10.1038/aps.2014.3

    Figure Lengend Snippet: Effect of the combination of LDM and CQ on apoptosis pathways. H460 cells were treated with both CQ and LDM (CQ pretreatment for 2 h) or none for 20 h. zVAD.fmk (100 μmol/L) were added before CQ and LDM for 1.5 h. (A) zVAD.fmk partly reversed the inhibitory effect of CQ combined with LDM on the proliferation of H460 cells. Mean±SD. n =3. (B) zVAD.fmk reduced the increasing expression of cleaved-PARP and cleaved-caspase 3 induced by CQ and LDM. (C) H460 cells were treated with CQ (50 μmol/L), LDM (0.5 nmol/L) and both (CQ pretreatment for 2 h) for 20 h. The influence of LDM and CQ (alone or both) on expression of p53, Bax, and Bcl-2. The expression levels of Bax and Bcl-2 were measured as the density by the Gel-Pro software, standardized by the density of β-actin.

    Article Snippet: Reagents Rabbit anti-caspase 3 antibody, rabbit anti-PARP antibody, rabbit anti-Bcl-2 antibody, rabbit anti-Bax antibody, and p53 antibody were all purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Expressing, Software

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

    Journal: Nature medicine

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

    doi: 10.1038/nm.4499

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

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

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

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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1004020

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

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

    Techniques: Western Blot, Transfection

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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1004020

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

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

    Techniques: Western Blot, Transfection, Double Knockout

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

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1004020

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

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

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

    Vorinostat and the BH3 mimetic ABT-737 cooperate to increase apoptosis in DLBCL cell lines. ( A ) SUDHL4, ( B ) Farage, ( C ) BJAB, ( D ) SUDHL6, and ( E ) RC-K8 cells were treated with ABT-737 in a dose-dependent manner with and without a sub-optimal level (0.5 µM) or a higher concentration (3 µM) of vorinostat. Whole-cell extracts were subjected to Western blotting to assess PARP cleavage and β-tubulin levels (as a normalizing control).

    Journal: PLoS ONE

    Article Title: The Sensitivity of Diffuse Large B-Cell Lymphoma Cell Lines to Histone Deacetylase Inhibitor-Induced Apoptosis Is Modulated by BCL-2 Family Protein Activity

    doi: 10.1371/journal.pone.0062822

    Figure Lengend Snippet: Vorinostat and the BH3 mimetic ABT-737 cooperate to increase apoptosis in DLBCL cell lines. ( A ) SUDHL4, ( B ) Farage, ( C ) BJAB, ( D ) SUDHL6, and ( E ) RC-K8 cells were treated with ABT-737 in a dose-dependent manner with and without a sub-optimal level (0.5 µM) or a higher concentration (3 µM) of vorinostat. Whole-cell extracts were subjected to Western blotting to assess PARP cleavage and β-tubulin levels (as a normalizing control).

    Article Snippet: Antibodies were as follows: BCL-2 (1∶500, BD Transduction Laboratories, San Jose, CA, USA, #610538), BCL-XL (1∶1000, Cell Signaling Technology, Danvers, MA, USA #2762), MCL-1 (1∶500, Santa Cruz Biotechnology, Santa Cruz, CA, USA, #819), BIM (1∶1000, Cell Signaling Technology, #2819), BAX (1∶500, Cell Signaling Technology), HRK (1∶1000, ProSci, Poway, CA, USA, #3771), BID (1∶1000, Santa Cruz Biotechnology, #11423), Caspase-3 (1∶500, Santa Cruz Biotechnology, #7148), Caspase-8 (1∶500, Santa Cruz Biotechnology, #7890), c-REL (1∶10000, kind gift of Nancy Rice, #265), p65 (1∶2000, kind gift of Nancy Rice, #1226), BCL6 (1∶1000, Cell Signaling Technology, #4242S), CD10 (1∶500, Santa Cruz Biotechnology, #58939), PARP (1∶500, Santa Cruz Biotechnology, #1750), and β-tubulin (1∶500, Santa Cruz Biotechnology, #9104).

    Techniques: Concentration Assay, Western Blot

    Trichostatin A and vorinostat induce PARP cleavage in a subset of DLBCL cell lines. The indicated DLBCL cell lines were treated with either 300 nM TSA (top panel) or 3 µM vorinostat (bottom panel) for 24 h and Western blotting was performed for PARP and β-tubulin (as a normalizing control). Note: in cases where cells undergo extensive PARP cleavage (e.g., SUDHL4, Pfeiffer, SUDHL2), tubulin can appear under-loaded (likely because tubulin also begins to be degraded in these dying cells). Cell lines used were GCB-like (BJAB, SUDHL4, SUDHL6, Pfeiffer, Farage, and SUDHL8) and ABC-like (RC-K8 and SUDHL2). Grey font indicates HDACi-sensitive cell lines; black font indicates HDACi-resistant cell line SUDHL6. The arrowhead indicates cleaved PARP.

    Journal: PLoS ONE

    Article Title: The Sensitivity of Diffuse Large B-Cell Lymphoma Cell Lines to Histone Deacetylase Inhibitor-Induced Apoptosis Is Modulated by BCL-2 Family Protein Activity

    doi: 10.1371/journal.pone.0062822

    Figure Lengend Snippet: Trichostatin A and vorinostat induce PARP cleavage in a subset of DLBCL cell lines. The indicated DLBCL cell lines were treated with either 300 nM TSA (top panel) or 3 µM vorinostat (bottom panel) for 24 h and Western blotting was performed for PARP and β-tubulin (as a normalizing control). Note: in cases where cells undergo extensive PARP cleavage (e.g., SUDHL4, Pfeiffer, SUDHL2), tubulin can appear under-loaded (likely because tubulin also begins to be degraded in these dying cells). Cell lines used were GCB-like (BJAB, SUDHL4, SUDHL6, Pfeiffer, Farage, and SUDHL8) and ABC-like (RC-K8 and SUDHL2). Grey font indicates HDACi-sensitive cell lines; black font indicates HDACi-resistant cell line SUDHL6. The arrowhead indicates cleaved PARP.

    Article Snippet: Antibodies were as follows: BCL-2 (1∶500, BD Transduction Laboratories, San Jose, CA, USA, #610538), BCL-XL (1∶1000, Cell Signaling Technology, Danvers, MA, USA #2762), MCL-1 (1∶500, Santa Cruz Biotechnology, Santa Cruz, CA, USA, #819), BIM (1∶1000, Cell Signaling Technology, #2819), BAX (1∶500, Cell Signaling Technology), HRK (1∶1000, ProSci, Poway, CA, USA, #3771), BID (1∶1000, Santa Cruz Biotechnology, #11423), Caspase-3 (1∶500, Santa Cruz Biotechnology, #7148), Caspase-8 (1∶500, Santa Cruz Biotechnology, #7890), c-REL (1∶10000, kind gift of Nancy Rice, #265), p65 (1∶2000, kind gift of Nancy Rice, #1226), BCL6 (1∶1000, Cell Signaling Technology, #4242S), CD10 (1∶500, Santa Cruz Biotechnology, #58939), PARP (1∶500, Santa Cruz Biotechnology, #1750), and β-tubulin (1∶500, Santa Cruz Biotechnology, #9104).

    Techniques: Western Blot

    Trichostatin A and vorinostat induce apoptosis and inhibit growth in a time- and dose-dependent manner. ( A ) SUDHL4 and SUDHL6 cells were treated with either 300 nM TSA or 3 µM vorinostat for the indicated times. Whole-cell extracts were made, and Western blotting for PARP and β-tubulin (as a normalizing control) was performed. ( B ) SUDHL4 and SUDHL6 cells were treated for 14 h with the indicated concentrations of either TSA or vorinostat. PARP cleavage and β-tubulin levels (as a normalizing control) were assessed by Western blotting of whole-cell extracts. ( C ) Relative caspase-3 activity was measured in cells treated for 14 h with either TSA or vorinostat as indicated. The values represent the average relative fluorescence of three independent assays as compared to untreated samples (1.0). Error bars indicate standard error. ( D ) SUDHL4 and SUDHL6 cells were treated with 3 µM vorinostat for 14 h, cells were stained with acridine orange and ethidium bromide, and counted for live (black bars), necrotic (grey bars), and apoptotic (white bars) cells (see Materials and Methods ). Each value is the average of three independent experiments and error bars indicate standard error. ( E ) Growth inhibition by vorinostat was assessed by treating SUDHL4 and SUDHL6 cells with increasing concentrations of vorinostat for 72 h, and cells were then counted using a hemocytometer. The relative numbers of cells are percentages as compared to cells grown for the same amount of time in the absence of vorinostat. The results are the averages of three separate treatment samples. Error bars indicate standard deviation. ( F ) SUDHL6 cells were treated with the indicated concentrations of vorinostat for 4 h. BIM and β-tubulin (as a normalizing control) were detected by Western blotting of whole-cell extracts. ( G ) SUDHL6 cells were treated for 24 h with the indicated concentrations of staurosporine. PARP cleavage and β-tubulin (as a normalizing control) were assessed by Western blotting of whole-cell extracts.

    Journal: PLoS ONE

    Article Title: The Sensitivity of Diffuse Large B-Cell Lymphoma Cell Lines to Histone Deacetylase Inhibitor-Induced Apoptosis Is Modulated by BCL-2 Family Protein Activity

    doi: 10.1371/journal.pone.0062822

    Figure Lengend Snippet: Trichostatin A and vorinostat induce apoptosis and inhibit growth in a time- and dose-dependent manner. ( A ) SUDHL4 and SUDHL6 cells were treated with either 300 nM TSA or 3 µM vorinostat for the indicated times. Whole-cell extracts were made, and Western blotting for PARP and β-tubulin (as a normalizing control) was performed. ( B ) SUDHL4 and SUDHL6 cells were treated for 14 h with the indicated concentrations of either TSA or vorinostat. PARP cleavage and β-tubulin levels (as a normalizing control) were assessed by Western blotting of whole-cell extracts. ( C ) Relative caspase-3 activity was measured in cells treated for 14 h with either TSA or vorinostat as indicated. The values represent the average relative fluorescence of three independent assays as compared to untreated samples (1.0). Error bars indicate standard error. ( D ) SUDHL4 and SUDHL6 cells were treated with 3 µM vorinostat for 14 h, cells were stained with acridine orange and ethidium bromide, and counted for live (black bars), necrotic (grey bars), and apoptotic (white bars) cells (see Materials and Methods ). Each value is the average of three independent experiments and error bars indicate standard error. ( E ) Growth inhibition by vorinostat was assessed by treating SUDHL4 and SUDHL6 cells with increasing concentrations of vorinostat for 72 h, and cells were then counted using a hemocytometer. The relative numbers of cells are percentages as compared to cells grown for the same amount of time in the absence of vorinostat. The results are the averages of three separate treatment samples. Error bars indicate standard deviation. ( F ) SUDHL6 cells were treated with the indicated concentrations of vorinostat for 4 h. BIM and β-tubulin (as a normalizing control) were detected by Western blotting of whole-cell extracts. ( G ) SUDHL6 cells were treated for 24 h with the indicated concentrations of staurosporine. PARP cleavage and β-tubulin (as a normalizing control) were assessed by Western blotting of whole-cell extracts.

    Article Snippet: Antibodies were as follows: BCL-2 (1∶500, BD Transduction Laboratories, San Jose, CA, USA, #610538), BCL-XL (1∶1000, Cell Signaling Technology, Danvers, MA, USA #2762), MCL-1 (1∶500, Santa Cruz Biotechnology, Santa Cruz, CA, USA, #819), BIM (1∶1000, Cell Signaling Technology, #2819), BAX (1∶500, Cell Signaling Technology), HRK (1∶1000, ProSci, Poway, CA, USA, #3771), BID (1∶1000, Santa Cruz Biotechnology, #11423), Caspase-3 (1∶500, Santa Cruz Biotechnology, #7148), Caspase-8 (1∶500, Santa Cruz Biotechnology, #7890), c-REL (1∶10000, kind gift of Nancy Rice, #265), p65 (1∶2000, kind gift of Nancy Rice, #1226), BCL6 (1∶1000, Cell Signaling Technology, #4242S), CD10 (1∶500, Santa Cruz Biotechnology, #58939), PARP (1∶500, Santa Cruz Biotechnology, #1750), and β-tubulin (1∶500, Santa Cruz Biotechnology, #9104).

    Techniques: Western Blot, Activity Assay, Fluorescence, Staining, Inhibition, Standard Deviation

    HDACi sensitivity is decreased by over-expression of BCL-2 or BCL-XL and increased by over-expression of BIM. Creation of four independent cell lines (SUDHL2 cells over-expressing BCL-2, SUDHL4 cells over-expressing BCL-XL, Farage cells over-expressing BCL-XL, and Farage cells over-expressing BIM) was confirmed by Western blotting for BCL-2, BCL-XL, and BIM ( A–D ) and compared to empty vector (–) transduced cells. β-tubulin was used as a normalizing control. ( A ) SUDHL2-BCL-2, ( B ) SUDHL4-BCL-XL, ( C ) Farage-BCL-XL, and ( D ) Farage-BIM cell lines were treated with either 300 nM TSA or 3 µM vorinostat for 24 h. Whole-cell extracts were made and Western blotting for PARP cleavage and β-tubulin (as a normalizing control) was performed. All cells over-expressing the protein of interest were compared to empty vector (–) transduced control cells.

    Journal: PLoS ONE

    Article Title: The Sensitivity of Diffuse Large B-Cell Lymphoma Cell Lines to Histone Deacetylase Inhibitor-Induced Apoptosis Is Modulated by BCL-2 Family Protein Activity

    doi: 10.1371/journal.pone.0062822

    Figure Lengend Snippet: HDACi sensitivity is decreased by over-expression of BCL-2 or BCL-XL and increased by over-expression of BIM. Creation of four independent cell lines (SUDHL2 cells over-expressing BCL-2, SUDHL4 cells over-expressing BCL-XL, Farage cells over-expressing BCL-XL, and Farage cells over-expressing BIM) was confirmed by Western blotting for BCL-2, BCL-XL, and BIM ( A–D ) and compared to empty vector (–) transduced cells. β-tubulin was used as a normalizing control. ( A ) SUDHL2-BCL-2, ( B ) SUDHL4-BCL-XL, ( C ) Farage-BCL-XL, and ( D ) Farage-BIM cell lines were treated with either 300 nM TSA or 3 µM vorinostat for 24 h. Whole-cell extracts were made and Western blotting for PARP cleavage and β-tubulin (as a normalizing control) was performed. All cells over-expressing the protein of interest were compared to empty vector (–) transduced control cells.

    Article Snippet: Antibodies were as follows: BCL-2 (1∶500, BD Transduction Laboratories, San Jose, CA, USA, #610538), BCL-XL (1∶1000, Cell Signaling Technology, Danvers, MA, USA #2762), MCL-1 (1∶500, Santa Cruz Biotechnology, Santa Cruz, CA, USA, #819), BIM (1∶1000, Cell Signaling Technology, #2819), BAX (1∶500, Cell Signaling Technology), HRK (1∶1000, ProSci, Poway, CA, USA, #3771), BID (1∶1000, Santa Cruz Biotechnology, #11423), Caspase-3 (1∶500, Santa Cruz Biotechnology, #7148), Caspase-8 (1∶500, Santa Cruz Biotechnology, #7890), c-REL (1∶10000, kind gift of Nancy Rice, #265), p65 (1∶2000, kind gift of Nancy Rice, #1226), BCL6 (1∶1000, Cell Signaling Technology, #4242S), CD10 (1∶500, Santa Cruz Biotechnology, #58939), PARP (1∶500, Santa Cruz Biotechnology, #1750), and β-tubulin (1∶500, Santa Cruz Biotechnology, #9104).

    Techniques: Over Expression, Expressing, Western Blot, Plasmid Preparation

    SUDHL4 cells selected for vorinostat resistance show decreased sensitivity to vorinostat-induced apoptosis and cell proliferation inhibition. ( A ) SUDHL4 and SUDHL4-VR cells were treated for 24 h with the indicated concentrations of vorinostat. PARP cleavage and β-tubulin (as a normalizing control) were assessed by Western blotting of whole-cell extracts. ( B ) Relative caspase-3 activity was measured in cells treated for 14 h with the indicated concentrations of vorinostat. The values represent the average relative fluorescence as compared to untreated samples (1.0) from three independent assays. Error bars indicate standard error. ( C ) Growth inhibition by vorinostat was assessed by treating SUDHL4-VR cells with increasing amounts of vorinostat for 72 h, and then counting cells using a hemocytometer. The relative numbers of cells are percentages as compared to cells incubated for the same amount of time in the absence of vorinostat. The results are the averages of three separate treatment samples. Error bars indicate standard deviation. ( D ) SUDHL4-VR cells were treated with the indicated concentrations of ABT-737 with and without a sub-optimal level (0.5 µM) of vorinostat. Whole-cell extracts were subjected to Western blotting for PARP cleavage and β-tubulin levels (as a normalizing control). ( E ) Relative caspase-3 activity was measured in cells treated for 14 h with TSA as indicated. The values represent the average relative fluorescence of three independent assays as compared to untreated samples. Error bars indicate standard error. ( F ) Cells were treated for 24 h with the indicated concentrations of staurosporine. PARP cleavage and β-tubulin (as a normalizing control) were assessed by Western blotting of whole-cell extracts. ( G ) Western blotting for the indicated BCL-2 family members in parental SUDHL4 cells and in two independent populations of SUDHL4-VR cells (1 and 2). β-tubulin was used as a protein loading control. The SUDHL4-VR(1) cell line was used for experiments in panels A–F.

    Journal: PLoS ONE

    Article Title: The Sensitivity of Diffuse Large B-Cell Lymphoma Cell Lines to Histone Deacetylase Inhibitor-Induced Apoptosis Is Modulated by BCL-2 Family Protein Activity

    doi: 10.1371/journal.pone.0062822

    Figure Lengend Snippet: SUDHL4 cells selected for vorinostat resistance show decreased sensitivity to vorinostat-induced apoptosis and cell proliferation inhibition. ( A ) SUDHL4 and SUDHL4-VR cells were treated for 24 h with the indicated concentrations of vorinostat. PARP cleavage and β-tubulin (as a normalizing control) were assessed by Western blotting of whole-cell extracts. ( B ) Relative caspase-3 activity was measured in cells treated for 14 h with the indicated concentrations of vorinostat. The values represent the average relative fluorescence as compared to untreated samples (1.0) from three independent assays. Error bars indicate standard error. ( C ) Growth inhibition by vorinostat was assessed by treating SUDHL4-VR cells with increasing amounts of vorinostat for 72 h, and then counting cells using a hemocytometer. The relative numbers of cells are percentages as compared to cells incubated for the same amount of time in the absence of vorinostat. The results are the averages of three separate treatment samples. Error bars indicate standard deviation. ( D ) SUDHL4-VR cells were treated with the indicated concentrations of ABT-737 with and without a sub-optimal level (0.5 µM) of vorinostat. Whole-cell extracts were subjected to Western blotting for PARP cleavage and β-tubulin levels (as a normalizing control). ( E ) Relative caspase-3 activity was measured in cells treated for 14 h with TSA as indicated. The values represent the average relative fluorescence of three independent assays as compared to untreated samples. Error bars indicate standard error. ( F ) Cells were treated for 24 h with the indicated concentrations of staurosporine. PARP cleavage and β-tubulin (as a normalizing control) were assessed by Western blotting of whole-cell extracts. ( G ) Western blotting for the indicated BCL-2 family members in parental SUDHL4 cells and in two independent populations of SUDHL4-VR cells (1 and 2). β-tubulin was used as a protein loading control. The SUDHL4-VR(1) cell line was used for experiments in panels A–F.

    Article Snippet: Antibodies were as follows: BCL-2 (1∶500, BD Transduction Laboratories, San Jose, CA, USA, #610538), BCL-XL (1∶1000, Cell Signaling Technology, Danvers, MA, USA #2762), MCL-1 (1∶500, Santa Cruz Biotechnology, Santa Cruz, CA, USA, #819), BIM (1∶1000, Cell Signaling Technology, #2819), BAX (1∶500, Cell Signaling Technology), HRK (1∶1000, ProSci, Poway, CA, USA, #3771), BID (1∶1000, Santa Cruz Biotechnology, #11423), Caspase-3 (1∶500, Santa Cruz Biotechnology, #7148), Caspase-8 (1∶500, Santa Cruz Biotechnology, #7890), c-REL (1∶10000, kind gift of Nancy Rice, #265), p65 (1∶2000, kind gift of Nancy Rice, #1226), BCL6 (1∶1000, Cell Signaling Technology, #4242S), CD10 (1∶500, Santa Cruz Biotechnology, #58939), PARP (1∶500, Santa Cruz Biotechnology, #1750), and β-tubulin (1∶500, Santa Cruz Biotechnology, #9104).

    Techniques: Inhibition, Western Blot, Activity Assay, Fluorescence, Incubation, Standard Deviation

    Effect of PARP-1 on diabetes-induced NF- k B/AP-1 binding at MMP-9 promoter in retinal microvessels: Binding of (a) NF- k B, and (b) AP-1 at MMP-9 promoter was measured in retinal microvessels using NF- k B and AP-1 antibodies. IgG was used as an antibody control (indicated as ^). (c) Retinal microvasculature from diabetic mice receiving PJ34 was quantified for MMP-9 expression by qPCR using 18S as the housekeeping gene. The values are represented as mean ± SD from 4–6 mice/group. Nor and Dia = C57BL/6J mice normal and diabetic respectively, and Dia+PJ34 = C57BL/6J diabetic mice administered with PJ34. * P

    Journal: Biochimica et biophysica acta

    Article Title: Role of PARP-1 as a novel transcriptional regulator of MMP-9 in diabetic retinopathy

    doi: 10.1016/j.bbadis.2017.04.024

    Figure Lengend Snippet: Effect of PARP-1 on diabetes-induced NF- k B/AP-1 binding at MMP-9 promoter in retinal microvessels: Binding of (a) NF- k B, and (b) AP-1 at MMP-9 promoter was measured in retinal microvessels using NF- k B and AP-1 antibodies. IgG was used as an antibody control (indicated as ^). (c) Retinal microvasculature from diabetic mice receiving PJ34 was quantified for MMP-9 expression by qPCR using 18S as the housekeeping gene. The values are represented as mean ± SD from 4–6 mice/group. Nor and Dia = C57BL/6J mice normal and diabetic respectively, and Dia+PJ34 = C57BL/6J diabetic mice administered with PJ34. * P

    Article Snippet: Chromatin immunoprecipitation (ChIP) was performed by immunoprecipitating 100–120μg crosslinked protein-DNA complex with antibodies against NF- k B (p65 subunit; ab7970, Abcam, Cambridge, MA), or AP-1 (c-Jun subunit; ab31419, Abcam), or PARP-1 (sc-7150, Santa Cruz Biotechnology).

    Techniques: Binding Assay, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction

    Regulation of PARP-1 and binding of NF- k B and AP-1 at the MMP-9 promoter: Endothelial cells incubated with PJ34 or PARP-1 siRNA, were analyzed for binding of (a) NF- k B, and (b) AP-1 at the proximal and the distal regions of MMP-9 promoter by ChIP technique. IgG was used as an antibody control (indicated as ^). Each measurement was made in duplicate in 3–4 samples/group. 5mM and 20mM=cells in 5mM or 20mM glucose; Mann = 20mM mannitol; 20+PJ34, si- P and C = cells incubated in 20mM glucose in the presence of PJ34, PARP-1 siRNA, and scrambled siRNA control respectively. * P

    Journal: Biochimica et biophysica acta

    Article Title: Role of PARP-1 as a novel transcriptional regulator of MMP-9 in diabetic retinopathy

    doi: 10.1016/j.bbadis.2017.04.024

    Figure Lengend Snippet: Regulation of PARP-1 and binding of NF- k B and AP-1 at the MMP-9 promoter: Endothelial cells incubated with PJ34 or PARP-1 siRNA, were analyzed for binding of (a) NF- k B, and (b) AP-1 at the proximal and the distal regions of MMP-9 promoter by ChIP technique. IgG was used as an antibody control (indicated as ^). Each measurement was made in duplicate in 3–4 samples/group. 5mM and 20mM=cells in 5mM or 20mM glucose; Mann = 20mM mannitol; 20+PJ34, si- P and C = cells incubated in 20mM glucose in the presence of PJ34, PARP-1 siRNA, and scrambled siRNA control respectively. * P

    Article Snippet: Chromatin immunoprecipitation (ChIP) was performed by immunoprecipitating 100–120μg crosslinked protein-DNA complex with antibodies against NF- k B (p65 subunit; ab7970, Abcam, Cambridge, MA), or AP-1 (c-Jun subunit; ab31419, Abcam), or PARP-1 (sc-7150, Santa Cruz Biotechnology).

    Techniques: Binding Assay, Incubation, Chromatin Immunoprecipitation

    Regulation of PARP-1 and its effect on mtDNA damage and cell apoptosis: Effect of PARP-1 regulation on (a) mtDNA-encoded Cytb expression was measured by qPCR using β-actin as the housekeeping gene, and (b) apoptosis by an ELISA kit for histone-associated-DNA-fragments in the cells incubated with PJ34 or PARP-1 siRNA, or scrambled siRNA control. The values are represented as mean ± SD from 3–4 samples/group. 5mM and 20mM=cells in 5mM or 20mM glucose; Mann = 20mM mannitol; 20+PJ34, si- P and C = cells incubated in 20mM glucose in the presence of PJ34, PARP-1 siRNA and scrambled siRNA control, respectively. * P

    Journal: Biochimica et biophysica acta

    Article Title: Role of PARP-1 as a novel transcriptional regulator of MMP-9 in diabetic retinopathy

    doi: 10.1016/j.bbadis.2017.04.024

    Figure Lengend Snippet: Regulation of PARP-1 and its effect on mtDNA damage and cell apoptosis: Effect of PARP-1 regulation on (a) mtDNA-encoded Cytb expression was measured by qPCR using β-actin as the housekeeping gene, and (b) apoptosis by an ELISA kit for histone-associated-DNA-fragments in the cells incubated with PJ34 or PARP-1 siRNA, or scrambled siRNA control. The values are represented as mean ± SD from 3–4 samples/group. 5mM and 20mM=cells in 5mM or 20mM glucose; Mann = 20mM mannitol; 20+PJ34, si- P and C = cells incubated in 20mM glucose in the presence of PJ34, PARP-1 siRNA and scrambled siRNA control, respectively. * P

    Article Snippet: Chromatin immunoprecipitation (ChIP) was performed by immunoprecipitating 100–120μg crosslinked protein-DNA complex with antibodies against NF- k B (p65 subunit; ab7970, Abcam, Cambridge, MA), or AP-1 (c-Jun subunit; ab31419, Abcam), or PARP-1 (sc-7150, Santa Cruz Biotechnology).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Incubation

    Effect of Sirt1 regulation on PARP-1 binding: Endothelial cells transfected with Sirt1 cDNA and incubated in 20mM glucose, were analyzed for the binding of PARP-1 at (a) NF- k B and (b) AP-1 binding regions of the MMP-9 promoter by ChIP technique. IgG was used as an antibody control (indicated as ^). (c) PARP-1 acetylation and its effect on the binding of NF- k B/AP-1 with PARP-1 were determined by immunoprecipitating total proteins using PARP-1 antibody, followed by western blotting for NF- k B/AP-1. The values are represented as mean ± SD from 3–4 samples/group. 5mM and 20mM = cells in 5mM or 20mM glucose; Mann = 20mM mannitol; 20+ St and 20+R = cells transfected with Sirt1 plasmids or transfection reagent alone respectively, and incubated in 20mM glucose. * P

    Journal: Biochimica et biophysica acta

    Article Title: Role of PARP-1 as a novel transcriptional regulator of MMP-9 in diabetic retinopathy

    doi: 10.1016/j.bbadis.2017.04.024

    Figure Lengend Snippet: Effect of Sirt1 regulation on PARP-1 binding: Endothelial cells transfected with Sirt1 cDNA and incubated in 20mM glucose, were analyzed for the binding of PARP-1 at (a) NF- k B and (b) AP-1 binding regions of the MMP-9 promoter by ChIP technique. IgG was used as an antibody control (indicated as ^). (c) PARP-1 acetylation and its effect on the binding of NF- k B/AP-1 with PARP-1 were determined by immunoprecipitating total proteins using PARP-1 antibody, followed by western blotting for NF- k B/AP-1. The values are represented as mean ± SD from 3–4 samples/group. 5mM and 20mM = cells in 5mM or 20mM glucose; Mann = 20mM mannitol; 20+ St and 20+R = cells transfected with Sirt1 plasmids or transfection reagent alone respectively, and incubated in 20mM glucose. * P

    Article Snippet: Chromatin immunoprecipitation (ChIP) was performed by immunoprecipitating 100–120μg crosslinked protein-DNA complex with antibodies against NF- k B (p65 subunit; ab7970, Abcam, Cambridge, MA), or AP-1 (c-Jun subunit; ab31419, Abcam), or PARP-1 (sc-7150, Santa Cruz Biotechnology).

    Techniques: Binding Assay, Transfection, Incubation, Chromatin Immunoprecipitation, Western Blot

    Effect of PARP-1 on the glucose- induced MMP-9 expression: Gene transcripts of (a) MMP-9 and (b) PARP-1 were quantified in the cells incubated in normal or high glucose in the presence of PJ34 or PARP-1 siRNA or scrambled siRNA by qPCR. β-actin was used as the housekeeping gene. (c) Nuclear localization of PARP-1 was determined by immunofluorescence using Alexa-Flour 488 (green) conjugated secondary antibody. The cover slips were mounted using DAPI-containing mounting medium (blue). Each measurement was made in duplicate in 4–5 samples in each group, and the values are represented as mean ± SD. 5mM and 20mM = cells in 5mM or 20mM glucose; Mann = 20mM mannitol; 20+PJ34, si- P and C = cells incubated with PJ34, PARP-1 siRNA, or scrambled siRNA respectively, and incubated in 20mM glucose. * P

    Journal: Biochimica et biophysica acta

    Article Title: Role of PARP-1 as a novel transcriptional regulator of MMP-9 in diabetic retinopathy

    doi: 10.1016/j.bbadis.2017.04.024

    Figure Lengend Snippet: Effect of PARP-1 on the glucose- induced MMP-9 expression: Gene transcripts of (a) MMP-9 and (b) PARP-1 were quantified in the cells incubated in normal or high glucose in the presence of PJ34 or PARP-1 siRNA or scrambled siRNA by qPCR. β-actin was used as the housekeeping gene. (c) Nuclear localization of PARP-1 was determined by immunofluorescence using Alexa-Flour 488 (green) conjugated secondary antibody. The cover slips were mounted using DAPI-containing mounting medium (blue). Each measurement was made in duplicate in 4–5 samples in each group, and the values are represented as mean ± SD. 5mM and 20mM = cells in 5mM or 20mM glucose; Mann = 20mM mannitol; 20+PJ34, si- P and C = cells incubated with PJ34, PARP-1 siRNA, or scrambled siRNA respectively, and incubated in 20mM glucose. * P

    Article Snippet: Chromatin immunoprecipitation (ChIP) was performed by immunoprecipitating 100–120μg crosslinked protein-DNA complex with antibodies against NF- k B (p65 subunit; ab7970, Abcam, Cambridge, MA), or AP-1 (c-Jun subunit; ab31419, Abcam), or PARP-1 (sc-7150, Santa Cruz Biotechnology).

    Techniques: Expressing, Incubation, Real-time Polymerase Chain Reaction, Immunofluorescence

    Combined treatment with carboplatin and thioridazine induced PSMA5 expression in an Nrf2-dependent manner. ( a ) AMC-HN4 cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for the indicated time periods. The protein expression levels of Nrf2 and actin were determined by western blotting. The level of actin was used as a loading control. ( b ) AMC-HN4 cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for the indicated time periods. After treatment, the nuclear extracts and cytosolic extracts were analyzed for Nrf2 and Ref-1 by western blotting as described in the Materials and Methods. Ref-1 was used as a marker for the nuclear fraction. ( c ) AMC-HN4 cells were transfected with an ARE-luciferase construct for 24 h, and the cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. After treatment, the cells were lysed and assayed for luciferase activity. ( d ) AMC-HN4 cells were transiently transfected with a control siRNA or Nrf2 siRNA. Twenty-four hours after transfection, the cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. The sub-G1 fraction was measured by flow cytometry as an indicator of the level of apoptosis. The protein expression levels of PARP, PSMA5, Nrf2, and actin were determined by western blotting. The values in c and d represent the mean±S.D. from three independent samples. * P

    Journal: Cell Death & Disease

    Article Title: Thioridazine enhances sensitivity to carboplatin in human head and neck cancer cells through downregulation of c-FLIP and Mcl-1 expression

    doi: 10.1038/cddis.2017.8

    Figure Lengend Snippet: Combined treatment with carboplatin and thioridazine induced PSMA5 expression in an Nrf2-dependent manner. ( a ) AMC-HN4 cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for the indicated time periods. The protein expression levels of Nrf2 and actin were determined by western blotting. The level of actin was used as a loading control. ( b ) AMC-HN4 cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for the indicated time periods. After treatment, the nuclear extracts and cytosolic extracts were analyzed for Nrf2 and Ref-1 by western blotting as described in the Materials and Methods. Ref-1 was used as a marker for the nuclear fraction. ( c ) AMC-HN4 cells were transfected with an ARE-luciferase construct for 24 h, and the cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. After treatment, the cells were lysed and assayed for luciferase activity. ( d ) AMC-HN4 cells were transiently transfected with a control siRNA or Nrf2 siRNA. Twenty-four hours after transfection, the cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. The sub-G1 fraction was measured by flow cytometry as an indicator of the level of apoptosis. The protein expression levels of PARP, PSMA5, Nrf2, and actin were determined by western blotting. The values in c and d represent the mean±S.D. from three independent samples. * P

    Article Snippet: The anti-Bcl-2, anti-Bcl-xL, anti-Mcl-1, anti-XIAP, anti-Nrf2, and anti-PARP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing, Western Blot, Marker, Transfection, Luciferase, Construct, Activity Assay, Flow Cytometry, Cytometry

    Reactive oxygen species has a critical role in carboplatin plus thioridazine-mediated PSMA5 expression. ( a ) AMC-HN4 cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for 6 h (left panel) or the indicated time periods (right panel), and the cells were then loaded with the H 2 DCF-DA fluorescent dye. The H 2 DCF-DA fluorescence intensity was detected by a fluorescence microscope (left panel) and flow cytometry (right panel). ( b ) AMC-HN4 cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for the indicated time periods. The protein expression levels of Prx-SO3 and actin were determined by western blotting. The level of actin was used as a loading control. ( c ) AMC-HN4 cells were pretreated with 5 mM NAC, 2 mM GEE, and 200 μ M trolox for 30 min and were then treated with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. After treatment, the nuclear extracts were analyzed for Nrf2 and Ref-1 by western blotting as described in the Materials and Methods. Ref-1 was used as a marker of the nuclear fraction. ( d ) AMC-HN4 cells were transfected with an ARE-luciferase construct for 24 h. The cells were pretreated with 5 mM NAC, 2 mM GEE, and 200 μ M trolox for 30 min and were then treated with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. After treatment, the cells were lysed and assayed for luciferase activity. ( e ) AMC-HN4 cells were transiently transfected with a plasmid harboring the luciferase gene under the control of the PSMA5/-277 promoter. After transfection, the cells were pretreated with 5 mM NAC, 2 mM GEE, and 200 μ M trolox for 30 min and were then treated with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. The luciferase activity was analyzed. ( f ) AMC-HN4 cells were pretreated with 5 mM NAC, 2 mM GEE, and 200 μ M trolox for 30 min and were then treated with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. The sub-G1 fraction was measured by flow cytometry. The protein expression levels of PARP, PSMA5, c-FLIP, Mcl-1, and actin were determined by western blotting. The level of actin was used as a loading control. The values in a , d , e and f represent the mean±S.D. from three independent samples. * P

    Journal: Cell Death & Disease

    Article Title: Thioridazine enhances sensitivity to carboplatin in human head and neck cancer cells through downregulation of c-FLIP and Mcl-1 expression

    doi: 10.1038/cddis.2017.8

    Figure Lengend Snippet: Reactive oxygen species has a critical role in carboplatin plus thioridazine-mediated PSMA5 expression. ( a ) AMC-HN4 cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for 6 h (left panel) or the indicated time periods (right panel), and the cells were then loaded with the H 2 DCF-DA fluorescent dye. The H 2 DCF-DA fluorescence intensity was detected by a fluorescence microscope (left panel) and flow cytometry (right panel). ( b ) AMC-HN4 cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for the indicated time periods. The protein expression levels of Prx-SO3 and actin were determined by western blotting. The level of actin was used as a loading control. ( c ) AMC-HN4 cells were pretreated with 5 mM NAC, 2 mM GEE, and 200 μ M trolox for 30 min and were then treated with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. After treatment, the nuclear extracts were analyzed for Nrf2 and Ref-1 by western blotting as described in the Materials and Methods. Ref-1 was used as a marker of the nuclear fraction. ( d ) AMC-HN4 cells were transfected with an ARE-luciferase construct for 24 h. The cells were pretreated with 5 mM NAC, 2 mM GEE, and 200 μ M trolox for 30 min and were then treated with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. After treatment, the cells were lysed and assayed for luciferase activity. ( e ) AMC-HN4 cells were transiently transfected with a plasmid harboring the luciferase gene under the control of the PSMA5/-277 promoter. After transfection, the cells were pretreated with 5 mM NAC, 2 mM GEE, and 200 μ M trolox for 30 min and were then treated with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. The luciferase activity was analyzed. ( f ) AMC-HN4 cells were pretreated with 5 mM NAC, 2 mM GEE, and 200 μ M trolox for 30 min and were then treated with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. The sub-G1 fraction was measured by flow cytometry. The protein expression levels of PARP, PSMA5, c-FLIP, Mcl-1, and actin were determined by western blotting. The level of actin was used as a loading control. The values in a , d , e and f represent the mean±S.D. from three independent samples. * P

    Article Snippet: The anti-Bcl-2, anti-Bcl-xL, anti-Mcl-1, anti-XIAP, anti-Nrf2, and anti-PARP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing, Fluorescence, Microscopy, Flow Cytometry, Cytometry, Western Blot, Marker, Transfection, Luciferase, Construct, Activity Assay, Plasmid Preparation

    Downregulation of c-FLIP and Mcl-1 expression by carboplatin plus thioridazine contributes to apoptosis. ( a and b ) AMC-HN4 cells were transiently transfected with pcDNA 3.1-c-FLIP ( a ) or pFLAG-CMV-4/Mcl-1 ( b ). Twenty-four hours after transfection, cells were treated with 200 nM carboplatin in the presence or absence of 10 μ M thioridazine for 24 h. The sub-G1 fraction was measured by flow cytometry. The protein expression levels of PARP, c-FLIP, Mcl-1, and actin were determined by western blotting. The level of actin was used as a loading control. ( c and d ) AMC-HN4 cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for the indicated time periods. The protein and mRNA expression levels of c-FLIP, Mcl-1, and actin were determined by western blotting and RT-PCR, respectively. The level of actin was used as a loading control. ( e ) AMC-HN4 cells were treated with or without 200 nM carboplatin plus 10 μ M thioridazine in the presence of 20 μ g/ml cyclohexamide (CHX) for the indicated time periods. The protein expression levels of c-FLIP, Mcl-1, and actin were determined by western blotting. The level of actin was used as a loading control. The band intensity of the c-FLIP and Mcl-1 protein was measured using ImageJ (public domain JAVA image-processing program ImageJ ( http://rsb.info.nih.gov/ij ). The values in a and b represent the mean±S.D. from three independent samples. * P

    Journal: Cell Death & Disease

    Article Title: Thioridazine enhances sensitivity to carboplatin in human head and neck cancer cells through downregulation of c-FLIP and Mcl-1 expression

    doi: 10.1038/cddis.2017.8

    Figure Lengend Snippet: Downregulation of c-FLIP and Mcl-1 expression by carboplatin plus thioridazine contributes to apoptosis. ( a and b ) AMC-HN4 cells were transiently transfected with pcDNA 3.1-c-FLIP ( a ) or pFLAG-CMV-4/Mcl-1 ( b ). Twenty-four hours after transfection, cells were treated with 200 nM carboplatin in the presence or absence of 10 μ M thioridazine for 24 h. The sub-G1 fraction was measured by flow cytometry. The protein expression levels of PARP, c-FLIP, Mcl-1, and actin were determined by western blotting. The level of actin was used as a loading control. ( c and d ) AMC-HN4 cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for the indicated time periods. The protein and mRNA expression levels of c-FLIP, Mcl-1, and actin were determined by western blotting and RT-PCR, respectively. The level of actin was used as a loading control. ( e ) AMC-HN4 cells were treated with or without 200 nM carboplatin plus 10 μ M thioridazine in the presence of 20 μ g/ml cyclohexamide (CHX) for the indicated time periods. The protein expression levels of c-FLIP, Mcl-1, and actin were determined by western blotting. The level of actin was used as a loading control. The band intensity of the c-FLIP and Mcl-1 protein was measured using ImageJ (public domain JAVA image-processing program ImageJ ( http://rsb.info.nih.gov/ij ). The values in a and b represent the mean±S.D. from three independent samples. * P

    Article Snippet: The anti-Bcl-2, anti-Bcl-xL, anti-Mcl-1, anti-XIAP, anti-Nrf2, and anti-PARP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing, Transfection, Flow Cytometry, Cytometry, Western Blot, Reverse Transcription Polymerase Chain Reaction

    Effect of carboplatin plus thioridazine on apoptosis in other cancer cells and in normal cells. ( a and b ) Breast cancer (MDA-MB231) and glioma (U87MG) cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. The sub-G1 fraction was measured by flow cytometry. The protein expression levels of PARP, Nrf2, PSMA5, c-FLIP, Mcl-1, and actin were determined by western blotting. The level of actin was used as a loading control. ( c and d ) AMC-HN4 cells, mesangial cells (MC), and normal human umbilical vein cells (EA.hy926) were treated with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. The cell morphology was examined using interference light microscopy ( c ). The sub-G1 fraction was measured by flow cytometry ( d ). The values in a , b , and d represent the mean±S.D. from three independent samples. * P

    Journal: Cell Death & Disease

    Article Title: Thioridazine enhances sensitivity to carboplatin in human head and neck cancer cells through downregulation of c-FLIP and Mcl-1 expression

    doi: 10.1038/cddis.2017.8

    Figure Lengend Snippet: Effect of carboplatin plus thioridazine on apoptosis in other cancer cells and in normal cells. ( a and b ) Breast cancer (MDA-MB231) and glioma (U87MG) cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. The sub-G1 fraction was measured by flow cytometry. The protein expression levels of PARP, Nrf2, PSMA5, c-FLIP, Mcl-1, and actin were determined by western blotting. The level of actin was used as a loading control. ( c and d ) AMC-HN4 cells, mesangial cells (MC), and normal human umbilical vein cells (EA.hy926) were treated with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. The cell morphology was examined using interference light microscopy ( c ). The sub-G1 fraction was measured by flow cytometry ( d ). The values in a , b , and d represent the mean±S.D. from three independent samples. * P

    Article Snippet: The anti-Bcl-2, anti-Bcl-xL, anti-Mcl-1, anti-XIAP, anti-Nrf2, and anti-PARP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Multiple Displacement Amplification, Flow Cytometry, Cytometry, Expressing, Western Blot, Light Microscopy

    Combined treatment with carboplatin and thioridazine upregulated the expression of PSMA5. ( a ) AMC-HN4 cells were pretreated with 0.5 μ M MG132 and 2.5 μ M lactacystin for 30 min and were then combined with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. The protein expression levels of c-FLIP, Mcl-1, and actin were determined by western blotting. The level of actin was used as a loading control. ( b ) AMC-HN4 cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for the indicated time periods. After treatment, the cells were lysed, and the proteasome activity was measured as described in the Materials and Methods section. ( c ) AMC-HN4 cells were treated with 200 nM carboplatin in the presence or absence of 10 μ M thioridazine for 24 h. The protein expression levels of PSMA5 and actin were determined by western blotting. The level of actin was used as a loading control. The band intensity of the PSMA5 protein was measured using ImageJ (public domain JAVA image-processing program ImageJ ( http://rsb.info.nih.gov/ij ). ( d ) AMC-HN4 cells were transiently transfected with a control siRNA or PSMA5 siRNA. Twenty-four hours after transfection, the cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. The sub-G1 fraction was measured by flow cytometry as an indicator of the level of apoptosis. The protein expression levels of PARP, c-FLIP, Mcl-1, PSMA5, and actin were determined by western blotting. The level of actin was used as a loading control. ( e ) AMC-HN4 cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for the indicated time periods. The protein and mRNA expression levels of PSMA5 and actin were determined by western blotting and RT-PCR, respectively. ( f ) AMC-HN4 cells were transiently transfected with a plasmid harboring the luciferase gene under the control of the PSMA5/-277 promoter. After transfection, the cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. The luciferase activity was analyzed. The values in b , d , and f represent the mean±S.D. from three independent samples. * P

    Journal: Cell Death & Disease

    Article Title: Thioridazine enhances sensitivity to carboplatin in human head and neck cancer cells through downregulation of c-FLIP and Mcl-1 expression

    doi: 10.1038/cddis.2017.8

    Figure Lengend Snippet: Combined treatment with carboplatin and thioridazine upregulated the expression of PSMA5. ( a ) AMC-HN4 cells were pretreated with 0.5 μ M MG132 and 2.5 μ M lactacystin for 30 min and were then combined with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. The protein expression levels of c-FLIP, Mcl-1, and actin were determined by western blotting. The level of actin was used as a loading control. ( b ) AMC-HN4 cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for the indicated time periods. After treatment, the cells were lysed, and the proteasome activity was measured as described in the Materials and Methods section. ( c ) AMC-HN4 cells were treated with 200 nM carboplatin in the presence or absence of 10 μ M thioridazine for 24 h. The protein expression levels of PSMA5 and actin were determined by western blotting. The level of actin was used as a loading control. The band intensity of the PSMA5 protein was measured using ImageJ (public domain JAVA image-processing program ImageJ ( http://rsb.info.nih.gov/ij ). ( d ) AMC-HN4 cells were transiently transfected with a control siRNA or PSMA5 siRNA. Twenty-four hours after transfection, the cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. The sub-G1 fraction was measured by flow cytometry as an indicator of the level of apoptosis. The protein expression levels of PARP, c-FLIP, Mcl-1, PSMA5, and actin were determined by western blotting. The level of actin was used as a loading control. ( e ) AMC-HN4 cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for the indicated time periods. The protein and mRNA expression levels of PSMA5 and actin were determined by western blotting and RT-PCR, respectively. ( f ) AMC-HN4 cells were transiently transfected with a plasmid harboring the luciferase gene under the control of the PSMA5/-277 promoter. After transfection, the cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. The luciferase activity was analyzed. The values in b , d , and f represent the mean±S.D. from three independent samples. * P

    Article Snippet: The anti-Bcl-2, anti-Bcl-xL, anti-Mcl-1, anti-XIAP, anti-Nrf2, and anti-PARP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing, Western Blot, Activity Assay, Transfection, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Luciferase

    Combined treatment with carboplatin and thioridazine induces apoptosis in human head and neck cancer (AMC-HN4) cells. ( a ) AMC-HN4 cells were treated with 200 nM carboplatin (Car) in the presence or absence of 10 μ M thioridazine (Thio) for 24 h. The sub-G1 fraction was measured by flow cytometry as an indicator of the level of apoptosis. The protein expression levels of PARP and actin were determined by western blot. The level of actin was used as a loading control. ( b ) Isoboles were obtained by plotting the combined concentrations of each drug required to produce 50% cell death. The straight line connecting the IC 50 values obtained for the two agents, when applied alone, corresponded to the addition of their independent effects. The values below this line indicate synergy, whereas the values above this line indicate antagonism. ( c – d ) AMC-HN4 cells were treated with 200 nM carboplatin in the presence or absence of 10 μ M thioridazine for 24 h. Condensation and fragmentation of the nuclei were detected by 4′,6′-diamidino-2-phenylindole staining ( c ). The cytoplasmic histone-associated DNA fragments were determined by a DNA fragmentation detection kit ( d ). Caspase activities were determined with colorimetric assays using caspase-3 (DEVDase) assay kits ( e ). ( f ) AMC-HN4 cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for 24 h in the presence or absence of 20 μ M z-VAD-fmk (z-VAD). The sub-G1 fraction was measured by flow cytometry. The protein expression levels of PARP, pro-caspase-3, cleaved caspase-3, and actin were determined by western blotting. The level of actin was used as a loading control. ( g ) AMC-HN4 cells were treated with 200 nM carboplatin in the presence or absence of 10 μ M thioridazine for 24 h. The protein expression levels of cIAP1, cIAP2, XIAP, c-FLIP, Mcl-1, Bcl-xL, Bcl-2, and actin were determined by western blotting. The level of actin was used as a loading control. The values in a , d , e , and f represent the mean±S.D. from three independent samples. * P

    Journal: Cell Death & Disease

    Article Title: Thioridazine enhances sensitivity to carboplatin in human head and neck cancer cells through downregulation of c-FLIP and Mcl-1 expression

    doi: 10.1038/cddis.2017.8

    Figure Lengend Snippet: Combined treatment with carboplatin and thioridazine induces apoptosis in human head and neck cancer (AMC-HN4) cells. ( a ) AMC-HN4 cells were treated with 200 nM carboplatin (Car) in the presence or absence of 10 μ M thioridazine (Thio) for 24 h. The sub-G1 fraction was measured by flow cytometry as an indicator of the level of apoptosis. The protein expression levels of PARP and actin were determined by western blot. The level of actin was used as a loading control. ( b ) Isoboles were obtained by plotting the combined concentrations of each drug required to produce 50% cell death. The straight line connecting the IC 50 values obtained for the two agents, when applied alone, corresponded to the addition of their independent effects. The values below this line indicate synergy, whereas the values above this line indicate antagonism. ( c – d ) AMC-HN4 cells were treated with 200 nM carboplatin in the presence or absence of 10 μ M thioridazine for 24 h. Condensation and fragmentation of the nuclei were detected by 4′,6′-diamidino-2-phenylindole staining ( c ). The cytoplasmic histone-associated DNA fragments were determined by a DNA fragmentation detection kit ( d ). Caspase activities were determined with colorimetric assays using caspase-3 (DEVDase) assay kits ( e ). ( f ) AMC-HN4 cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for 24 h in the presence or absence of 20 μ M z-VAD-fmk (z-VAD). The sub-G1 fraction was measured by flow cytometry. The protein expression levels of PARP, pro-caspase-3, cleaved caspase-3, and actin were determined by western blotting. The level of actin was used as a loading control. ( g ) AMC-HN4 cells were treated with 200 nM carboplatin in the presence or absence of 10 μ M thioridazine for 24 h. The protein expression levels of cIAP1, cIAP2, XIAP, c-FLIP, Mcl-1, Bcl-xL, Bcl-2, and actin were determined by western blotting. The level of actin was used as a loading control. The values in a , d , e , and f represent the mean±S.D. from three independent samples. * P

    Article Snippet: The anti-Bcl-2, anti-Bcl-xL, anti-Mcl-1, anti-XIAP, anti-Nrf2, and anti-PARP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Flow Cytometry, Cytometry, Expressing, Western Blot, Staining

    Mitochondrial reactive oxygen species are important for carboplatin plus thioridazine-induced apoptosis. ( a ) AMC-HN4 cells were pretreated with 100 μ M apocynin, 50 nM DPI, and 20 nM rotenone for 30 min followed by stimulation with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. The sub-G1 fraction was measured by flow cytometry. The protein expression levels of PARP, Nrf2, PSMA5, c-FLIP, Mcl-1, and actin were determined by western blotting. The level of actin was used as a loading control. ( b ) AMC-HN4 cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for 3 h (left panel) or the indicated time periods (right panel). The cells were then loaded with the Mitosox Red fluorescent dye. The Mitosox Red fluorescence intensity was detected by a fluorescence microscope (left panel) and flow cytometry (right panel). ( c ) AMC-HN4 cells were treated with 200 nM carboplatin in the presence or absence of 10 μ M thioridazine for 6 h. The cells were then loaded with the H 2 DCF-DA fluorescent dye. The H 2 DCF-DA fluorescence intensity was detected by flow cytometry. ( d ) AMC-HN4 cells were pretreated with the indicated concentrations of Mito-TEMPO and were then added with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. The sub-G1 fraction was measured by flow cytometry. The protein expression levels of PARP, Nrf2, PSMA5, c-FLIP, Mcl-1, and actin were determined by western blotting. The level of actin was used as a loading control. The values in a , b , c , and d represent the mean±S.D. from three independent samples. * P

    Journal: Cell Death & Disease

    Article Title: Thioridazine enhances sensitivity to carboplatin in human head and neck cancer cells through downregulation of c-FLIP and Mcl-1 expression

    doi: 10.1038/cddis.2017.8

    Figure Lengend Snippet: Mitochondrial reactive oxygen species are important for carboplatin plus thioridazine-induced apoptosis. ( a ) AMC-HN4 cells were pretreated with 100 μ M apocynin, 50 nM DPI, and 20 nM rotenone for 30 min followed by stimulation with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. The sub-G1 fraction was measured by flow cytometry. The protein expression levels of PARP, Nrf2, PSMA5, c-FLIP, Mcl-1, and actin were determined by western blotting. The level of actin was used as a loading control. ( b ) AMC-HN4 cells were treated with 200 nM carboplatin plus 10 μ M thioridazine for 3 h (left panel) or the indicated time periods (right panel). The cells were then loaded with the Mitosox Red fluorescent dye. The Mitosox Red fluorescence intensity was detected by a fluorescence microscope (left panel) and flow cytometry (right panel). ( c ) AMC-HN4 cells were treated with 200 nM carboplatin in the presence or absence of 10 μ M thioridazine for 6 h. The cells were then loaded with the H 2 DCF-DA fluorescent dye. The H 2 DCF-DA fluorescence intensity was detected by flow cytometry. ( d ) AMC-HN4 cells were pretreated with the indicated concentrations of Mito-TEMPO and were then added with 200 nM carboplatin plus 10 μ M thioridazine for 24 h. The sub-G1 fraction was measured by flow cytometry. The protein expression levels of PARP, Nrf2, PSMA5, c-FLIP, Mcl-1, and actin were determined by western blotting. The level of actin was used as a loading control. The values in a , b , c , and d represent the mean±S.D. from three independent samples. * P

    Article Snippet: The anti-Bcl-2, anti-Bcl-xL, anti-Mcl-1, anti-XIAP, anti-Nrf2, and anti-PARP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Flow Cytometry, Cytometry, Expressing, Western Blot, Fluorescence, Microscopy

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

    Journal: Oncotarget

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

    doi: 10.18632/oncotarget.11065

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

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

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

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

    Journal: Oncotarget

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

    doi: 10.18632/oncotarget.11065

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

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

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

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

    Journal: Oncotarget

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

    doi: 10.18632/oncotarget.11065

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

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

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

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

    Journal: Oncotarget

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

    doi: 10.18632/oncotarget.11065

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

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

    Techniques: Multiple Displacement Amplification

    ROCK I does not activate caspases. Wild-type and lamin A/C null MEFs expressing ROCK I:ER or kinase dead KD:ER were left untreated or treated with 1 μM 4-HT, either in the absence or presence of z-VAD-fmk as indicated. As a positive control for caspase activation, cells were treated with 25 ng/ml TNFα plus 10 μg/ml CHX. Western blotting revealed that 4-HT treatment of KD:ER- or ROCK I:ER-expressing wild-type and lamin A/C null fibroblasts did not induce cleavage of lamin B1 (A) or PARP (B) although each was cleaved in response to TNFα treatment. (C) Western blotting for ERK2 reflects protein levels across samples.

    Journal: The Journal of Cell Biology

    Article Title: Actin-myosin-based contraction is responsible for apoptotic nuclear disintegration

    doi: 10.1083/jcb.200409049

    Figure Lengend Snippet: ROCK I does not activate caspases. Wild-type and lamin A/C null MEFs expressing ROCK I:ER or kinase dead KD:ER were left untreated or treated with 1 μM 4-HT, either in the absence or presence of z-VAD-fmk as indicated. As a positive control for caspase activation, cells were treated with 25 ng/ml TNFα plus 10 μg/ml CHX. Western blotting revealed that 4-HT treatment of KD:ER- or ROCK I:ER-expressing wild-type and lamin A/C null fibroblasts did not induce cleavage of lamin B1 (A) or PARP (B) although each was cleaved in response to TNFα treatment. (C) Western blotting for ERK2 reflects protein levels across samples.

    Article Snippet: Blots were probed with antibodies against lamins B1 and A/C (Santa Cruz Biotechnology, Inc.), PARP (BD Biosciences), β-tubulin (Amersham Biosciences), GFP (CLONTECH Laboratories, Inc.), phospho-MLC (Thr18/Ser19), cleaved caspase 3 (Asp 175; Cell Signaling), MLC (Sigma-Aldrich), phospho-MYPT1 (Thr696; Upstate Biotechnology), MYPT1 (Covance), ROCK I (BD Biosciences), phospho-Ezrin (Thr567; Cell Signaling), Ezrin (Upstate Biotechnology), ERα (Santa Cruz Biotechnology, Inc.), phospho-LIMK1(Thr508)/LIMK2(Thr505) (Cell Signaling), LIMK1 (Cell Signaling), ERK2 (provided by C.J.

    Techniques: Expressing, Positive Control, Activation Assay, Western Blot

    Apoptotic nuclear breakdown is blocked following actin filament disruption. (A) Actin filaments were apparent in untreated NIH 3T3 cells (left), but cell morphology and actin filaments were profoundly affected by treatment with 2 μM cytochalasin D (right). Bars, 20 μm. (B) TEM of cell pretreated with 2 μM cytochalasin D to disrupt actin structures before induction of apoptosis, showing intact nucleus. Bar, 2 μm. (C) Activation of Caspase 3 and cleavage of ROCK I (D) and PARP (E) were not affected by cytochalasin treatment and disruption of the actin cytoskeleton before the induction of apoptosis. (F) Blotting for β-tubulin indicates equal loading across lanes.

    Journal: The Journal of Cell Biology

    Article Title: Actin-myosin-based contraction is responsible for apoptotic nuclear disintegration

    doi: 10.1083/jcb.200409049

    Figure Lengend Snippet: Apoptotic nuclear breakdown is blocked following actin filament disruption. (A) Actin filaments were apparent in untreated NIH 3T3 cells (left), but cell morphology and actin filaments were profoundly affected by treatment with 2 μM cytochalasin D (right). Bars, 20 μm. (B) TEM of cell pretreated with 2 μM cytochalasin D to disrupt actin structures before induction of apoptosis, showing intact nucleus. Bar, 2 μm. (C) Activation of Caspase 3 and cleavage of ROCK I (D) and PARP (E) were not affected by cytochalasin treatment and disruption of the actin cytoskeleton before the induction of apoptosis. (F) Blotting for β-tubulin indicates equal loading across lanes.

    Article Snippet: Blots were probed with antibodies against lamins B1 and A/C (Santa Cruz Biotechnology, Inc.), PARP (BD Biosciences), β-tubulin (Amersham Biosciences), GFP (CLONTECH Laboratories, Inc.), phospho-MLC (Thr18/Ser19), cleaved caspase 3 (Asp 175; Cell Signaling), MLC (Sigma-Aldrich), phospho-MYPT1 (Thr696; Upstate Biotechnology), MYPT1 (Covance), ROCK I (BD Biosciences), phospho-Ezrin (Thr567; Cell Signaling), Ezrin (Upstate Biotechnology), ERα (Santa Cruz Biotechnology, Inc.), phospho-LIMK1(Thr508)/LIMK2(Thr505) (Cell Signaling), LIMK1 (Cell Signaling), ERK2 (provided by C.J.

    Techniques: Transmission Electron Microscopy, Activation Assay

    Degradation of nuclear lamins A/C and B1, LAP2α, Nup153, and PARP during apoptosis is unaffected by ROCK inhibition. NIH 3T3 fibroblasts were left untreated or treated with 10 μM Y-27632 to inhibit ROCK activity or 50 μM z-VAD-fmk to inhibit caspase activity as indicated. Apoptosis was induced with TNFα/CHX where indicated. Lysates were prepared and Western blotted with antibodies against lamins A and C (A), lamin B1 (B), LAP-2α (C), Nup-153 (D), and PARP (E). Equal loading was verified with an antibody against β-tubulin.

    Journal: The Journal of Cell Biology

    Article Title: Actin-myosin-based contraction is responsible for apoptotic nuclear disintegration

    doi: 10.1083/jcb.200409049

    Figure Lengend Snippet: Degradation of nuclear lamins A/C and B1, LAP2α, Nup153, and PARP during apoptosis is unaffected by ROCK inhibition. NIH 3T3 fibroblasts were left untreated or treated with 10 μM Y-27632 to inhibit ROCK activity or 50 μM z-VAD-fmk to inhibit caspase activity as indicated. Apoptosis was induced with TNFα/CHX where indicated. Lysates were prepared and Western blotted with antibodies against lamins A and C (A), lamin B1 (B), LAP-2α (C), Nup-153 (D), and PARP (E). Equal loading was verified with an antibody against β-tubulin.

    Article Snippet: Blots were probed with antibodies against lamins B1 and A/C (Santa Cruz Biotechnology, Inc.), PARP (BD Biosciences), β-tubulin (Amersham Biosciences), GFP (CLONTECH Laboratories, Inc.), phospho-MLC (Thr18/Ser19), cleaved caspase 3 (Asp 175; Cell Signaling), MLC (Sigma-Aldrich), phospho-MYPT1 (Thr696; Upstate Biotechnology), MYPT1 (Covance), ROCK I (BD Biosciences), phospho-Ezrin (Thr567; Cell Signaling), Ezrin (Upstate Biotechnology), ERα (Santa Cruz Biotechnology, Inc.), phospho-LIMK1(Thr508)/LIMK2(Thr505) (Cell Signaling), LIMK1 (Cell Signaling), ERK2 (provided by C.J.

    Techniques: Inhibition, Activity Assay, Western Blot

    Atg7 knockdown enhanced PARP cleavage in CPT- or STS-treated, but not in PDT-treated, MCF-7 cells. C: Control. MCF-7 cells (Atg7 + and Atg7 − ) were exposed to 200 nM Pc 4 and 200 mJ/cm 2 red light then further incubated for 24 h or treated with 2 µM CPT for 24 h (A). Cells were also incubated in 1 µM sTs for 6 h (B). At the end of the incubation, whole cell lysates were subjected to electrophoresis and analyzed by western blot for the presence of PARP cleavage, and the levels of LC3 and Atg7.

    Journal: Autophagy

    Article Title: Atg7 deficiency increases resistance of MCF-7 human breast cancer cells to photodynamic therapy

    doi:

    Figure Lengend Snippet: Atg7 knockdown enhanced PARP cleavage in CPT- or STS-treated, but not in PDT-treated, MCF-7 cells. C: Control. MCF-7 cells (Atg7 + and Atg7 − ) were exposed to 200 nM Pc 4 and 200 mJ/cm 2 red light then further incubated for 24 h or treated with 2 µM CPT for 24 h (A). Cells were also incubated in 1 µM sTs for 6 h (B). At the end of the incubation, whole cell lysates were subjected to electrophoresis and analyzed by western blot for the presence of PARP cleavage, and the levels of LC3 and Atg7.

    Article Snippet: Equivalent amounts of protein were loaded onto polyacrylamide gels, subjected to electrophoresis, transferred to a PVDF membrane and incubated with anti-LC3 antibody (a kind gift from Dr. T. Yoshimori, National Institute of Genetics, Japan), anti-Atg7 antibody (ProSci Inc., 3617), anti-PARP antibody (BD Pharmingen, 556362), anti-Beclin 1 antibody (BD Transduction Laboratories, 612113), or anti-actin antibody (NeoMarkers, MS-1295).

    Techniques: Cycling Probe Technology, Incubation, Electrophoresis, Western Blot

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

    Journal: Journal of Cellular and Molecular Medicine

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

    doi: 10.1111/j.1582-4934.2008.00259.x

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

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

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

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

    Journal: Journal of Cellular and Molecular Medicine

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

    doi: 10.1111/j.1582-4934.2008.00259.x

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

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

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

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

    Journal: Journal of Cellular and Molecular Medicine

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

    doi: 10.1111/j.1582-4934.2008.00259.x

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

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

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

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

    Journal: Journal of Cellular and Molecular Medicine

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

    doi: 10.1111/j.1582-4934.2008.00259.x

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

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

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

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

    Journal: Journal of Cellular and Molecular Medicine

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

    doi: 10.1111/j.1582-4934.2008.00259.x

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

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

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

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

    Journal: Journal of Cellular and Molecular Medicine

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

    doi: 10.1111/j.1582-4934.2008.00259.x

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

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

    Techniques: Binding Assay, Mass Spectrometry

    The function of miR-142-3p in sorafenib resistance is dependent on the regulation of ATG5 and ATG16L1. a MTT assay was performed to measure the sensitivity of SMMC-7721 cells co-transfected with miR-142-3p mimics and ATG5 or ATG16L1 to sorafenib. b Colony-formation assay was employed to determine the proliferation ability of SMMC-7721 cells co-transfected with miR-142-3p mimics and ATG5 or ATG16L1. c Flow cytometric assay was employed to measure the apoptosis rate in SMMC-7721 cells co-transfected with miR-142-3p mimics and ATG5 or ATG16L1. d Western blotting was utilized to assess the levels of the apoptosis-related proteins c-caspase-3 and c-PARP and autophagy-related proteins in SMMC-7721 cells co-transfected with miR-142-3p mimics and ATG5 or ATG16L1. All data are presented as the mean ± S.D. from three independent experiments. The p -values represent comparisons between groups (** p

    Journal: Cell Death & Disease

    Article Title: PU.1/microRNA-142-3p targets ATG5/ATG16L1 to inactivate autophagy and sensitize hepatocellular carcinoma cells to sorafenib

    doi: 10.1038/s41419-018-0344-0

    Figure Lengend Snippet: The function of miR-142-3p in sorafenib resistance is dependent on the regulation of ATG5 and ATG16L1. a MTT assay was performed to measure the sensitivity of SMMC-7721 cells co-transfected with miR-142-3p mimics and ATG5 or ATG16L1 to sorafenib. b Colony-formation assay was employed to determine the proliferation ability of SMMC-7721 cells co-transfected with miR-142-3p mimics and ATG5 or ATG16L1. c Flow cytometric assay was employed to measure the apoptosis rate in SMMC-7721 cells co-transfected with miR-142-3p mimics and ATG5 or ATG16L1. d Western blotting was utilized to assess the levels of the apoptosis-related proteins c-caspase-3 and c-PARP and autophagy-related proteins in SMMC-7721 cells co-transfected with miR-142-3p mimics and ATG5 or ATG16L1. All data are presented as the mean ± S.D. from three independent experiments. The p -values represent comparisons between groups (** p

    Article Snippet: The membranes were blocked with 5% nonfat dry milk in TBST for 1 h, and then incubated with primary antibody anti-PU.1 (Abcam, Cat#:ab76543), anti-LC3B (Abcam, Cat#:ab48394), anti-p62 (Abcam, Cat#:ab56416), anti-caspase3 (Abcam, Cat#:ab13586), anti-c-caspase3 (Abcam, Cat#:ab2302), anti-PARP (Abcam, Cat#:ab32138), anti-c-PARP (Abcam, Cat#:ab32064), anti-ATG5 (Abcam, Cat#:ab227132), anti-ATG16L1 (Abcam, Cat#:ab188642) and anti-GAPDH (Abcam, Cat#:ab8245) overnight at 4 °C before subsequent incubation with second antibody (Cell Signaling Technology) for 1 h at 37 °C.

    Techniques: MTT Assay, Transfection, Colony Assay, Flow Cytometry, Western Blot

    Induction of apoptosis by alkylaminoguaiazulenes in HSC-2 cells. Near-confluent HSC-2 cells were incubated for 24 h with the indicated concentrations of alkylaminoguaiazulenes and expression of cleaved products of poly ADP-ribose polymerase (PARP) and caspase-3 was visualized by western blot analysis. Each sample contains 0.1% DMSO. Act.D., Actinomycin D (1 μM), was used as a positive control for apoptosis.

    Journal: In Vivo

    Article Title: In Vitro Antitumor Activity of Alkylaminoguaiazulenes

    doi: 10.21873/invivo.112273

    Figure Lengend Snippet: Induction of apoptosis by alkylaminoguaiazulenes in HSC-2 cells. Near-confluent HSC-2 cells were incubated for 24 h with the indicated concentrations of alkylaminoguaiazulenes and expression of cleaved products of poly ADP-ribose polymerase (PARP) and caspase-3 was visualized by western blot analysis. Each sample contains 0.1% DMSO. Act.D., Actinomycin D (1 μM), was used as a positive control for apoptosis.

    Article Snippet: Antibodies against cleaved caspase-3 (Cell Signaling Technology Inc., Beverly, MD, USA), poly ADP-ribose polymerase (PARP; Cell Signaling Technology Inc.) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Trevigen, Gaithersburg, MD, USA) were used as primary antibodies.

    Techniques: Incubation, Expressing, Western Blot, Activated Clotting Time Assay, Positive Control

    ZEB1 promotes EMT . A-C . Immunoblots. PANC-1 cells were pre-treated with TGF-β1 for two days and then transfected twice (day 0 and day 2) with ZEB1 siRNAs in the continued presence of TGF-β1. Four days after the initial transfection, cells were harvested. A . By up-regulating epithelial proteins such as E-cadherin and CAR, knockdown of ZEB1 antagonizes TGF-β-induced EMT in PANC-1 cells. Similarly, silencing of ZEB1 expression in MDA-MB-231 cells up-regulates E-cadherin and CAR, and down-regulates the mesenchymal marker fibronectin. B . PANC-1 cells were treated with TGF-β1, and harvested at the indicated time-points for analysis of the total protein fractions. C . PANC-1 cells were treated with TGF-β1 and subjected to cell fractionation. Abbreviations: C, TGF-β1 solvent control [4 mM HCl/0.1% (v/w) BSA]; UT, untransfected; Ctrl #1, siControl ON-TARGETplus Non-targeting siRNA #1 (Dharmacon); Ctrl #2, firefly luciferase-targeting siRNA; ZEB1 siRNA #1/#2, ZEB1-targeting siRNAs. Ctrl #2 and ZEB1 siRNA sequences are provided in Additional file 1 (Table S3). Chinese Hamster Ovary cells stably expressing human CAR (CHO+), or vector (CHO-) [ 9 ]. Loading controls are shown as β-actin, β-tubulin, GAPDH and PARP signals, with GAPDH as a cytoplasmic, and PARP as a nuclear marker.

    Journal: Molecular Cancer

    Article Title: ZEB1 limits adenoviral infectability by transcriptionally repressing the Coxsackie virus and Adenovirus Receptor

    doi: 10.1186/1476-4598-10-91

    Figure Lengend Snippet: ZEB1 promotes EMT . A-C . Immunoblots. PANC-1 cells were pre-treated with TGF-β1 for two days and then transfected twice (day 0 and day 2) with ZEB1 siRNAs in the continued presence of TGF-β1. Four days after the initial transfection, cells were harvested. A . By up-regulating epithelial proteins such as E-cadherin and CAR, knockdown of ZEB1 antagonizes TGF-β-induced EMT in PANC-1 cells. Similarly, silencing of ZEB1 expression in MDA-MB-231 cells up-regulates E-cadherin and CAR, and down-regulates the mesenchymal marker fibronectin. B . PANC-1 cells were treated with TGF-β1, and harvested at the indicated time-points for analysis of the total protein fractions. C . PANC-1 cells were treated with TGF-β1 and subjected to cell fractionation. Abbreviations: C, TGF-β1 solvent control [4 mM HCl/0.1% (v/w) BSA]; UT, untransfected; Ctrl #1, siControl ON-TARGETplus Non-targeting siRNA #1 (Dharmacon); Ctrl #2, firefly luciferase-targeting siRNA; ZEB1 siRNA #1/#2, ZEB1-targeting siRNAs. Ctrl #2 and ZEB1 siRNA sequences are provided in Additional file 1 (Table S3). Chinese Hamster Ovary cells stably expressing human CAR (CHO+), or vector (CHO-) [ 9 ]. Loading controls are shown as β-actin, β-tubulin, GAPDH and PARP signals, with GAPDH as a cytoplasmic, and PARP as a nuclear marker.

    Article Snippet: Immunoblotting and cell fractionation Antibodies used include rabbit anti-phospho-Smad2 (Ser465/467, Cell Signaling Technology, Inc.), goat anti-ZEB1 (E-20, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), mouse anti-β-tubulin (Sigma-Aldrich), mouse anti-PARP (clone C2-10, Pharmingen/BD Biosciences, San Jose, CA, USA), mouse anti-GAPDH-Peroxidase Conjugate (Sigma-Aldrich), and mouse anti-Myc Tag (clone 4A6; Upstate/Millipore, Charlottesville, VA, USA).

    Techniques: Western Blot, Transfection, Expressing, Multiple Displacement Amplification, Marker, Cell Fractionation, Luciferase, Stable Transfection, Plasmid Preparation