Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) Identification of RNF168-associated proteins. A representative SDS–polyacrylamide gel electrophoresis of Flag-RNF168-associated proteins. Flag-tagged RNF168 was transfected in HEK293T cells and pull-down analysis was performed 48 h later. Protein bands were detected by silver staining. Protein bands were identified by mass spectrometry analysis following in-gel protease digestion. ( b ) HEK293T cells were transfected as indicated with HA-tagged RNF168 and Flag-TOP2α expression vectors. Cells were lysed and IP was performed using anti-Flag antibody. The resulting precipitates were subjected to IB analysis with the indicated antibodies. WCL, whole-cell lysate. ( c ) TOP2α, RNF168 and IgG (control) immunoprecipitates from HEK293T cells were examined by IB as indicated. ( b , c ) Data are representative of three independent experiments. ( d ) Cells treated with EdU were used for detection of localization patterns of TOP2α (Alexa Fluor 488) and RNF168 (Alexa Fluor 594) using confocal microscopy. Scale bar, 20 μm.

Article Snippet: Recombinant RNF168 (ref. ; 0.5–2 μg), recombinant TOP2α (5 μg, Topogen), UBE1 (0.1 μg; E1), UBE2E2 (0.2 μg; E2), Ub (5 μg) and ATP (5 mM) were mixed and the reactions were incubated at 30 °C for 90 min in buffer containing 50 mM Tris/HCl (pH 7.5), 5 mM MgCl2, and 1 mM DTT, and examined by IB.

Techniques: Polyacrylamide Gel Electrophoresis, Transfection, Silver Staining, Mass Spectrometry, Expressing, Control, Confocal Microscopy

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) Representative agarose gel of in vitro kinetoplast DNA-based decatenation assays performed for 10 min with different amount of nuclear extracts from WT and Rnf168 −/− MEFs. Catenated and decatenated kDNA were separated by electrophoresis using 1% agarose gel. IB show Top2α's level in the total nuclear extracts used for this assay. ( b ) Representative data of the mitotic inhibition assay of decatenation G2 checkpoint in WT and Rnf168 −/− primary MEFs. Cells were treated with DMSO or ICRF-193 for 15 min and then incubated in culture media for an additional 2 h. The fraction of mitotic cells (pHH3 + ) was determined by flow cytometry. ( c ) Bar graphs represent the mean inhibition of mitotic index 2 h post ICRF-193 treatment of passage 1 primary MEFs (% pHH3 + cells post ICRF-193 treatment compared with DMSO-treated controls). * P <0.05. ( d ) Bar graphs represent the mean inhibition of mitotic index 2 h post ICRF-193 treatment of RIDDLE cells reconstituted with either HA-RNF168 or HA-empty vector as in c . * P <0.05. ( e ) Analysis of decatenation G2 checkpoint of Rnf168 −/− and WT 3T3 MEFs using the mitotic entry assay. Percentage of pHH3 + cells is shown at the indicated time post-treatment with colcemid in the presence of DMSO or ICRF-193. * P <0.05; Rnf168 −/− MEFs compared with WT MEFs 6 h post-ICRF-193 treatment. ( f ) Bar graphs represent the mean fraction of pHH3 + WT and Rnf168 −/− MEFs evading G2 arrest 6 h post-treatment with ICRF-193 compared with DMSO-treated cells as in e . * P <0.05. ( g ) Mitotic entry assay of decatenation G2 checkpoint in human RIDDLE cells reconstituted with HA-empty vector or HA-RNF168. Percentage of pHH3 + cells is shown at the indicated times post-treatment with colcemid with or without ICRF-193. * P <0.05, RIDDLE cells reconstituted with HA-empty vector compared with RIDDLE cells reconstituted with HA-RNF168 at 6 h post-ICRF-193 treatment. ( h ) Bar graphs represent the mean fraction of RIDDLE cells (reconstituted with HA-empty vector compared with those reconstituted with HA-RNF168) evading G2 arrest 6 h post ICRF-193 treatment compared with DMSO-treated controls as in f . * P <0.05. Three independent experiments in triplicates unless specified. Error bars in c , d , f and h represent mean±s.e.m.

Article Snippet: Recombinant RNF168 (ref. ; 0.5–2 μg), recombinant TOP2α (5 μg, Topogen), UBE1 (0.1 μg; E1), UBE2E2 (0.2 μg; E2), Ub (5 μg) and ATP (5 mM) were mixed and the reactions were incubated at 30 °C for 90 min in buffer containing 50 mM Tris/HCl (pH 7.5), 5 mM MgCl2, and 1 mM DTT, and examined by IB.

Techniques: Agarose Gel Electrophoresis, In Vitro, Electrophoresis, Inhibition, Incubation, Flow Cytometry, Plasmid Preparation

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) RIDDLE cells reconstituted with HA-RNF168 or HA-empty vector, and control HA-RNF168-reconstituted RIDDLE cells with TOP2α knock down were lysed and WCL subjected to IP with anti-TOP2α or IgG (control) antibodies. Immunoprecipitates were blotted with the indicated antibodies to detect ubiquitylated TOP2α. ( b ) Human breast cancer cell lines T47D and MDA-MB-231 knocked down for RNF168 (Sh.RNF168) and their control expressing ShRNA control (sh.Ctr) were examined for their level of ubiquitylated TOP2α as in a . ( c ) Rnf168 −/− , Brca1 −/− and WT MEFs were lysed and subjected to IP with anti-Top2α or IgG (control) antibodies. IPs from WCL were blotted with the indicated antibodies. ( d ) HEK293T cells were transfected with RNF168 (WT or mutant Rnf168-C21S), Flag-TOP2α and HA-Ub vectors as indicated. WCL were subjected to IP with anti-Flag, and IB analysis was performed using anti-HA antibody to detect ubiquitylated Flag-TOP2α. ( e ) Nuclear extracts from RIDDLE cells reconstituted with HA-RNF168 or HA-empty vector were subjected to IP with anti-TOP2α or IgG (control) antibodies. Immunoprecipitates were blotted with the indicated antibodies against K63- and K48-Ub linkages. ( f ) In vitro ubiquitylation of recombinant TOP2α in the presence of recombinant RNF168 (500 ng for lane 5, 1 μg for lanes 2, 3 and 6 and 2 μg for lane7), UBE1 (E1), UBE2E2 (E2) and Ub. Nuc, nuclear extract; WCL, whole-cell lysate.

Article Snippet: Recombinant RNF168 (ref. ; 0.5–2 μg), recombinant TOP2α (5 μg, Topogen), UBE1 (0.1 μg; E1), UBE2E2 (0.2 μg; E2), Ub (5 μg) and ATP (5 mM) were mixed and the reactions were incubated at 30 °C for 90 min in buffer containing 50 mM Tris/HCl (pH 7.5), 5 mM MgCl2, and 1 mM DTT, and examined by IB.

Techniques: Plasmid Preparation, Control, Knockdown, Expressing, shRNA, Transfection, Mutagenesis, In Vitro, Recombinant

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) WT and Rnf168 −/− MEFs, and Top2α knockdown control MEFs were fixed and stained with DAPI. Representative cells with defective chromosome segregation, as indicated by chromosome bridges (arrow head) and micronuclei (arrow) are shown. ( b ) Histograms show quantification of cells with chromosome bridges or micronuclei (mean±s.e.m., n =3). * P <0.05. ( c ) Histograms show the fraction of RIDDLE cells reconstituted with HA-RNF168 or HA-empty vector that display micronuclei (mean±s.e.m., n =3). * P <0.05. ( d ; upper panels) Representative metaphase spreads showing undercondensed chromosomes in WT MEFs 24 h post ICRF-193 treatment (positive control), and in Rnf168 −/− DMSO-treated MEFs. ( d , lower panels) Representative metaphase spreads of DMSO-treated Rnf168 −/− MEFs complemented with RNF168-WT (WT) or the E3 ligase deficient RNF168-C21S (C21S). ( e ) Histograms show quantification of abnormal metaphase spreads with entangled or undercondensed chromosomes from the indicated cells (mean±s.e.m., n =3). * P <0.05 compared with WT MEFs. ( f ) Histograms present the mean fraction of MEFs evading ICRF-193-induced G2 arrest as compared with DMSO-treated controls (mean±s.e.m., n =3). Data are shown for WT MEFs, mock infected Rnf168 −/− MEFs and Rnf168 −/− MEFs complemented with RNF168-WT or RNF168-C21S. 3T3 MEFs were used for these experiments. Data shown are for 6 h post-treatment with colcemid±4 μM ICRF-193. * P <0.05 compared with WT MEFs. ( g ) Clonogenic assay was used to determine sensitivity to ICRF-193 of mock infected WT and Rnf168 −/− MEFs, as well as Rnf168 −/− MEFs complemented with RNF168 (WT or C21S mutant). Data are presented as the mean±s.e.m. ( n =4). * P <0.05 compared with WT MEFs. Scale bar, 20 μm.

Article Snippet: Recombinant RNF168 (ref. ; 0.5–2 μg), recombinant TOP2α (5 μg, Topogen), UBE1 (0.1 μg; E1), UBE2E2 (0.2 μg; E2), Ub (5 μg) and ATP (5 mM) were mixed and the reactions were incubated at 30 °C for 90 min in buffer containing 50 mM Tris/HCl (pH 7.5), 5 mM MgCl2, and 1 mM DTT, and examined by IB.

Techniques: Knockdown, Control, Staining, Plasmid Preparation, Positive Control, Infection, Clonogenic Assay, Mutagenesis

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) Nuclear (Nuc) and chromatin (Chr) fractions prepared from WT, Rnf168 −/− and Brca1 −/− MEFs were analysed by IB for chromatin occupancy of Top2α. Additional IBs were performed with the indicated antibodies as controls. H4, histone H4. ( b ) Nuclear and chromatin fractions were prepared from the human breast cancer cell lines T47D and MDA-MB-231 knocked down for RNF168 (Sh.RNF168) and their controls (Sh.Ctr) and analysed by IB for the chromatin occupancy of TOP2α as in a . ( c ) HEK293T were transfected with Flag-TOP2α along with RNF168 (+) or empty vector (−) and their nuclear and chromatin fractions were prepared and examined by IB using the indicated antibodies. ( d ) A representative agarose gel showing decatenation activity of soluble nuclear and chromatin extracts from 2 WT and 2 Rnf168 −/− MEFs. In vitro kinetoplast DNA-based decatenation assay was performed for 20 min with different amounts of nuclear and chromatin extracts, and catenated and decatenated kDNA were separated by electrophoresis. IB using anti-Top2α was performed to show the level of Top2α present in each sample.

Article Snippet: Recombinant RNF168 (ref. ; 0.5–2 μg), recombinant TOP2α (5 μg, Topogen), UBE1 (0.1 μg; E1), UBE2E2 (0.2 μg; E2), Ub (5 μg) and ATP (5 mM) were mixed and the reactions were incubated at 30 °C for 90 min in buffer containing 50 mM Tris/HCl (pH 7.5), 5 mM MgCl2, and 1 mM DTT, and examined by IB.

Techniques: Transfection, Plasmid Preparation, Agarose Gel Electrophoresis, Activity Assay, In Vitro, Electrophoresis

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a , b ) HEK293T cells were transfected with Flag-RNF168 and HA-USP10 vectors ( a ) or Flag-TOP2α and HA-USP10 vectors ( b ) as indicated. Cells were lysed and IP was performed using anti-Flag and anti-HA antibodies. The resulting precipitates were subjected to IB analysis with the indicated antibodies. WCL, whole-cell lysate. ( c ) HEK293T cells were transfected with Flag-TOP2α, RNF168, HA-USP10 and Myc-Ub vectors, as indicated. IP using anti-Flag and WCL was subjected to anti-Ub IB analysis to detect TOP2α ubiquitylation. ( d ) WT MEFs with knockdown of Usp10 (Sh1 and Sh2) and WT controls were examined for the level of Top2α ubiquitylation. Top2α was immunoprecipitated from whole-cell extracts and examined by IB for its ubiquitylation level using anti-Ub. IP using IgG was used as a control. The indicated antibodies were used for IB. ( e ) HEK293T cells were transfected with Flag-TOP2α with or without RNF168, HA-Ub, USP10 and USP10-C424A as indicated. TOP2α chromatin occupancy in these cells was examined by IB using anti-Flag antibodies and chromatin fractions (Chr). IB analysis of the chromatin fractions is also shown for the indicated antibodies. ( f ) A simplified model of RNF168-mediated regulation of TOP2α ubiquitylation and decatenation function.

Article Snippet: Recombinant RNF168 (ref. ; 0.5–2 μg), recombinant TOP2α (5 μg, Topogen), UBE1 (0.1 μg; E1), UBE2E2 (0.2 μg; E2), Ub (5 μg) and ATP (5 mM) were mixed and the reactions were incubated at 30 °C for 90 min in buffer containing 50 mM Tris/HCl (pH 7.5), 5 mM MgCl2, and 1 mM DTT, and examined by IB.

Techniques: Transfection, Knockdown, Immunoprecipitation, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: MFTZ-1 reduces constitutive and inducible HIF-1α accumulation and VEGF secretion independent of its topoisomerase II inhibition

doi: 10.1111/j.1582-4934.2009.00822.x

Figure Lengend Snippet: MFTZ-1 decreases VEGF secretion in MDA-MB-231 cells. (A) MFTZ-1 inhibited hypoxia-induced VEGF gene transcription (upper) and VEGF protein secretion (lower) in MDA-MB-231 cells. The cells were treated with the indicated concentrations of MFTZ-1 at hypoxia for 6 hrs (VEGF transcription) or 16 hrs (VEGF secretion). Then total RNA was isolated for VEGF transcription analyses by real-time PCR; or cell supernatants were collected and measured for VEGF levels by ELISA. (B) HIF-1α was silenced with 80 nM specific HIF-1α siRNA (siHIF-1α) in both mRNA (lower) and protein (upper) levels. HIF-1α protein was detected by Western blotting in MDA-MB-231 cells transfected with negative control (NC) or siHIF-1α duplexes. Twenty-four hours after transfection, cells were treated with or without MG132 for 4 hrs. Real-time RT-PCR was done to analyse the level of HIF-1α mRNA in MDA-MB-231 cells 24 hrs after transfection. (C) Specific down-regulation of HIF-1α with 80 nM siHIF-1α did not affect the effects of MFTZ-1 on VEGF transcription (upper) and secretion (lower) at normoxia. MDA-MB-231 cells were transfected with siHIF-1α or NC for 40 hrs. In the last 16 hrs of the transfection, the cells were treated with or without MFTZ-1. Then real-time RT-PCR and ELISA assays were done separately for detection of VEGF transcription and secretion. (D) Silencing HIF-1α (80 nM siHIF-1α) removed the hypoxia-induced increment of VEGF secretion, and MFTZ-1 reduced VEGF secretion of the siHIF-1α-transfected MDA-MB-231 cells at hypoxia. Cells were transfected with siHIF-1α for 40 hrs. In the last 16 hrs of the transfection, the cells were treated with or without MFTZ-1 at hypoxia. Then ELISA assays were done for detection of VEGF secretion. The data represent three independent experiments. Student’s t-tests were performed to compare hypoxic control with normoxic control and each drug treatment with the hypoxic control in (A), to compare all other treatment with control in (B), (C) and (D). The significance was indicated as * for P < 0.05 and ** for P < 0.01.

Article Snippet: siRNA duplexes for Top2α RNA (sense: GGUAUUCCUGUUGUUGAAC) [ ] and the HIF-1α siRNA (sense 5′-CUG AUG ACC AGC AAC UUG AdTdT-3′) [ ] were synthesized by Shanghai GenePharma Co., Ltd. Transfection was done with Oligofectamine (Invitrogen) according to the manufacturer’s instructions.

Techniques: Isolation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Negative Control, Quantitative RT-PCR, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: MFTZ-1 reduces constitutive and inducible HIF-1α accumulation and VEGF secretion independent of its topoisomerase II inhibition

doi: 10.1111/j.1582-4934.2009.00822.x

Figure Lengend Snippet: MFTZ-1 reduces HIF-1α and inhibits tube formation in a Top2-independent manner. (A) Transfection efficiency of siRNA targeting Top2α (100 nM) in MDA-MB-231 cells. The cells were collected for Western blotting 36 hrs after transfection. (B) Silencing Top2α did not affect the MFTZ-1-mediated decrease in HIF-1α protein. After transfection with mock or Top2α siRNA for 36 hrs, the cells were moved to a hypoxia incubator and continued to be incubated with or without MFTZ-1(0.2, 1 μM) for 4 hrs before Western blotting was done. (C) MFTZ-1 reduced HIF-1α protein accumulation in HL60 and functional Top2-defective HL60/MX2. Cells were treated with indicated concentration of MFTZ-1 at hypoxia for 6 hrs. Then the cells were lysed and detected for HIF-1α and β-Actin by Western blotting. Data shown were representative of three independent experiments. (D) Transfection efficiency of Top2α (100 nM) siRNA in HUVEC cells. The cells were collected for Western blotting 36 hrs after transfection. (E) Silencing Top2α did not affect the MFTZ-1-mediated inhibition of HUVEC tube formation. After transfection with mock or Top2α siRNAs for 36 hrs, HUVEC cells (1.2 × 10 4 ) in M199 medium with or without 1 μM MFTZ-1 were added to matrigel and continued to be incubated for 8 hrs at 37°C. Capillary networks were photographed. Data shown were representative of three independent experiments. (F) The separable activities of MFTZ-1 on angiogenesis and Top2.

Article Snippet: siRNA duplexes for Top2α RNA (sense: GGUAUUCCUGUUGUUGAAC) [ ] and the HIF-1α siRNA (sense 5′-CUG AUG ACC AGC AAC UUG AdTdT-3′) [ ] were synthesized by Shanghai GenePharma Co., Ltd. Transfection was done with Oligofectamine (Invitrogen) according to the manufacturer’s instructions.

Techniques: Transfection, Western Blot, Incubation, Functional Assay, Concentration Assay, Inhibition

Journal: Nucleic Acids Research

Article Title: Ciprofloxacin impairs mitochondrial DNA replication initiation through inhibition of Topoisomerase 2

doi: 10.1093/nar/gky793

Figure Lengend Snippet: Topoisomerase 2α and 2β co-localize with replicating mitochondrial DNA nucleoids. ( A ) Detection of topoisomerases in mitochondrial extracts of human and mouse cell lines by Western blot. Human cell lines: HEK293T and HeLa. Mouse cells: proliferating C2C12 myoblasts and differentiated C2C12 myotubes, mouse embryonic fibroblasts of wildtype and Top2β (–/–) knockout mice. ATAD3 is used as loading reference. Note that the used polyclonal antibodies might detect mouse and human proteins with different efficiencies. ( B ) Detection of topoisomerase Top2β, Top1mt and Top3α in mitochondrial extracts of various mouse tissues by Western blot. TFAM is used as indicator of mtDNA content, while TOMM20 serves as mitochondrial loading control. ( C ) Localization of mitochondrial topoisomerases in floatation gradients of mitochondrial extracts from HEK293T cells. The pellet fraction after digitonin treatment contains inner membrane-associated proteins and nucleoids, while the supernatant fraction contains soluble nucleoids and proteins without membrane-association. Top2α and Top2b co-localize well with the membrane-attached, replicating mtDNA nucleoids characterized by the presence of the mitochondrial helicase TWNK. Also the mitochondrial topoisomerases Top3a and Top1mt partially co-localize with this fraction, but a larger proportion is found in other fractions. ( D ) Mitochondrially targeted Top2β co-localizes with replicating nucleoids. HeLa cells transiently transfected with an expression vector for myc-tagged, mitochondrially targeted Top2β were immuno-stained with a monoclonal antibody against the myc-tag (green) and a polyclonal antibody against mtSSB (red) as a marker for mtDNA replication.

Article Snippet: Rabbit-anti-Top2α , 1:1000 , Abgent , AP9248b.

Techniques: Western Blot, Knock-Out, Transfection, Expressing, Plasmid Preparation, Staining, Marker

Journal: Nucleic Acids Research

Article Title: Ciprofloxacin impairs mitochondrial DNA replication initiation through inhibition of Topoisomerase 2

doi: 10.1093/nar/gky793

Figure Lengend Snippet: Knockdown of Top2β, but not Top2α affects mtDNA topology and replication. ( A ) mtDNA topology in HeLa cells after 3 days knockdown of Top2α, Top2β or Top2α and β simultaneously. The left panel shows the different topological forms of mtDNA. In vitro treatment with TopIV relaxes supercoiled mtDNA and removes catenanes, while it does not modify mtDNA multimers. The right panel shows the mtDNA topology after a 3 day siRNA knockdown of Top2a and b. Loss of Top2β, but not Top2α leads to accumulation of supercoiled mtDNA. Protein levels of Top2α and β after knockdown were determined by Western blot to confirm the reduction oin Top2 protein levels. ( B ) mtDNA copy number in HeLa cells after 3 days knockdown of Top2α, Top2β or Top2α and β simultaneously. Loss of Top2β alone or in combination with Top2α knockdown leads to a significant decrease of mtDNA ( n = 3, ANOVA/Holm inference, * indicates P < 0.05, ns non-significant).

Article Snippet: Rabbit-anti-Top2α , 1:1000 , Abgent , AP9248b.

Techniques: In Vitro, Western Blot

Journal: Nucleic Acids Research

Article Title: Ciprofloxacin impairs mitochondrial DNA replication initiation through inhibition of Topoisomerase 2

doi: 10.1093/nar/gky793

Figure Lengend Snippet:

Article Snippet: Rabbit-anti-Top2α , 1:1000 , Abgent , AP9248b.

Techniques: