Journal: Scientific Reports

Article Title: Dynamic behavior of DNA topoisomerase IIβ in response to DNA double-strand breaks

doi: 10.1038/s41598-018-28690-6

Figure Lengend Snippet: Suppression of Topo IIβ recruitment to DSB sites by ICRF-187 and ICRF-193. ( A ) Immunofluorescent staining of endogenous Topo IIβ and phosphorylated DNA-PKcs in the presence and absence of Topo II inhibitors. HeLa cells were pretreated with DMSO, 20 µM ICRF-187, 20 µM ICRF-193, or 40 µM merbarone for 1 h and subsequently subjected to laser microirradiation. After 10 min incubation at 37 °C, cells were coimmunostained for Topo IIβ and pS2056 of DNA-PKcs. Representative images are shown. ( B ) Schematic diagram of domains and amino acid substitutions in human Topo IIβ. Topo IIβ is composed of an ATPase domain, a catalytic core domain, and a C-terminal domain (CTD). The amino acid substitutions G180I and L185F are considered to confer insensitivity of Topo IIβ to the ICRF inhibitors. The Y821S substitution is inferred to impair the topoisomerase activity. ( C ) Recruitment of EGFP-Topo IIβ mutants to DSB sites in the presence of ICRF-187. EGFP-Topo IIβ mutants were transiently expressed in HeLa cells. After pretreatment with DMSO or ICRF-187 for 1 h, cells were subjected to laser microirradiation followed by live cell imaging. Representative images at 30 sec after DSB induction are shown.

Article Snippet: ICRF-193 and merbarone were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Staining, Incubation, Activity Assay, Live Cell Imaging, Size-exclusion Chromatography

Journal: Nucleic Acids Research

Article Title: Transient dsDNA breaks during pre-replication complex assembly

doi: 10.1093/nar/gkp617

Figure Lengend Snippet: Origin association of replication initiator proteins during G 1 phase. ( A ) Immunostaining showing the nuclear expression of ORC2, ORC4, Cdt1 and Cdc45 upon release from M phase. ORC2, known to be stably expressed throughout the cell cycle, represents the loading control. ( B–C ) ChIP assay showing the association of ORC2 (i), ORC4 (ii), Cdt1 (iii) and Cdc45 (iv) with the hOrs8 (B) and lamin B2 (C) origins with (grey bars) or without (black bars) merbarone treatment. Abundance of immunoprecipitated origin DNA is expressed in molecules per 2 × 10 7 cells and the error bars represent three experiments and 1 SD. The horizontal bars represent background DNA immunoprecipitated by NRS.

Article Snippet: Treatments with the topo II inhibitor merbarone (100 μM) (Calbiochem) and the DNA-PK inhibitor NU-7026 (10 μM) (Calbiochem) were performed for 1 h at 37°C.

Techniques: Immunostaining, Expressing, Stable Transfection, Chromatin Immunoprecipitation, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: Transient dsDNA breaks during pre-replication complex assembly

doi: 10.1093/nar/gkp617

Figure Lengend Snippet: DNA-break formation during G 1 phase. ( A ) Schematic diagram explaining the three different scenarios of the DNA-break labelling assay. No break formation (left) should lead to background levels of DNA immunoprecipitated by the anti-biotin antibody. In the case of a break in proximity to the origin, but outside of the core region (middle), increased amounts of the PCR products upstream (Set 1), downstream (Set 2) and flanking (Ori) the origin region should be produced, while an intra-origin break (right) should result in increased Set 1 and Set 2, but decreased Ori products. ( B ) Generation of putative DNA breaks in hOrs8 (i) and lamin B2 (ii) during G 1 phase. Encircled are the two consecutive DNA breaks formed at the two chromosomal loci, two intra-origin breaks in hOrs8, and one inter-origin and one intra-origin in lamin B2. Values are expressed as ratios of the individual DNA abundances at each timepoint to the maximal abundance observed during the course of the assay, following normalization with the non-origin containing regions. Error bars represent 1 SD of three separate experiments. ( C ) Effect of merbarone on DNA-break formation at the hOrs8 (i) and lamin B2 (ii) replication origins. Error bars represent 1 SD of three separate experiments. ( D ) DNA-break labelling assay at the hOrs8 (i) and lamin B2 (ii) replication origins following NU-7026 treatment. Error bars represent 1 SD of three separate experiments.

Article Snippet: Treatments with the topo II inhibitor merbarone (100 μM) (Calbiochem) and the DNA-PK inhibitor NU-7026 (10 μM) (Calbiochem) were performed for 1 h at 37°C.

Techniques: Immunoprecipitation, Polymerase Chain Reaction, Produced

Journal: Molecules

Article Title: In Vitro Action of Flavonoids in the Canine Malignant Histiocytic Cell Line DH82

doi: 10.3390/molecules181215448

Figure Lengend Snippet: Chemical structure of flavonoids and topoisomerase inhibitors. ( A ) General structure of flavonoids; ( B ) general structure of chalcones; ( C ) quercetin; ( D ) myricetin; ( E ) fisetin; ( F ) epigallocatechin gallate (EGCG); ( G ) genistein; ( H ) luteolin; ( I ) apigenin; ( J ) trans- chalcone; ( L ) 4-methoxychalcone; ( M ) etoposide; ( N ) merbarone.

Article Snippet: Materials The topoisomerase inhibitors merbarone, etoposide, quercetin, trans -chalcone, and 4-methoxychalcone were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques:

Journal: Molecules

Article Title: In Vitro Action of Flavonoids in the Canine Malignant Histiocytic Cell Line DH82

doi: 10.3390/molecules181215448

Figure Lengend Snippet: Inhibition of the viability of DH82 cells induced by flavonoids and topoisomerase after 24, 48 and 72 h of treatment. ( A ) Quercetin; ( B ) myricetin; ( C ) fisetin; ( D ) epigallocatechin gallate (EGCG); ( E ) genistein; ( F ) luteolin; ( G ) apigenin; ( H ) trans-chalcone; ( I ) 4-methoxychalcone; ( J ) etoposide; ( L ) merbarone. Symbols represent the mean of at least three independent experiments and the error bar indicates the standard deviation. Different letters indicate statistically significant results ( p

Article Snippet: Materials The topoisomerase inhibitors merbarone, etoposide, quercetin, trans -chalcone, and 4-methoxychalcone were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Inhibition, Standard Deviation

Journal: Molecules

Article Title: In Vitro Action of Flavonoids in the Canine Malignant Histiocytic Cell Line DH82

doi: 10.3390/molecules181215448

Figure Lengend Snippet: Effect of flavonoids and topoisomerase inhibitors on the expression of topoisomerase IIa and IIb genes. The cells were treated with flavonoids at a concentration of 12.5 µg/mL for 6 h. The concentration of etoposide and merbarone was 2.5 µg/mL. The results are expressed as the mean ± standard error of at least three independent experiments. Different letters indicate statistically significant results ( p

Article Snippet: Materials The topoisomerase inhibitors merbarone, etoposide, quercetin, trans -chalcone, and 4-methoxychalcone were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Expressing, Concentration Assay

Journal: Nature Communications

Article Title: Topoisomerase II? promotes activation of RNA polymerase I transcription by facilitating pre-initiation complex formation

doi: 10.1038/ncomms2599

Figure Lengend Snippet: Effects of Top2 inhibition on Pol I transcription. ( a ) Schematic of pulse-chase labelling of cells with 3 H-uridine to determine effects of Top2 inhibition on rRNA synthesis by Pol I (used in b and c ) ( b ) Pre-rRNA (47/45S), 28S and 18S rRNA transcript levels in actively growing U2OS cells were unaffected by treatment with Top2 inhibitor merbarone (Me) for up to 15 h. 3 H-uridine was added 1 h after Me; non-radioactive uridine was added after an additional 2 h. Total RNA was extracted after 0, 4 and 12 h, separated by formaldehyde agarose gel electrophoresis and transferred to membrane. Newly synthesized pre-rRNA and rRNA transcripts were detected by autoradiography (representative experiment; upper panel); total 18S and 28S rRNAs were detected by ethidium bromide staining (lower panel). Relative efficiencies of rRNA synthesis and transcript processing are shown (bar graphs: −Me, dark-blue bars;+Me, light-red bars) for 47S/45S pre-rRNA (top), 28S rRNA (middle) and 18S rRNA (bottom), expressed as percentage of highest value (set at 100%). s.d. is shown, n =3; * P

Article Snippet: For Top2 inhibition, Top2 poison etoposide (100 μM final concentration; Merck) or catalytic inhibitors ICRF-193 (50 μM) and merbarone (100 μM; Merck) were added (except ).

Techniques: Inhibition, Pulse Chase, Agarose Gel Electrophoresis, Synthesized, Autoradiography, Staining

Journal: Nature Communications

Article Title: Topoisomerase II? promotes activation of RNA polymerase I transcription by facilitating pre-initiation complex formation

doi: 10.1038/ncomms2599

Figure Lengend Snippet: Top2α facilitates formation of PICs in Pol I transcription activation. ( a , b ) Top2 inhibition reduces activation of Pol I transcription in serum-refed cells. H1299 cells (p53 null) ( a ) or U2OS cells ( b ) were serum-starved for 20 h and (starting at –1 h) incubated with Top2 inhibitors etoposide (Et) or merbarone (Me), respectively, or without inhibitors, for 1 h. At 0 h, serum was added to the cells. RNA was extracted 15, 30, 60 and 90 min after serum addition. Pre-rRNA levels were analysed by S1 nuclease protection (complementary assay, 3 H-uridine labelling of RNA, Supplementary Fig. S6a,b ); s.d. is shown. ( c ) Top2α depletion reduces activation of Pol I transcription in serum-refed cells. HTETOP cells were incubated for 48 h +/− Tet, with serum starvation for the last 20 h. Serum was added back and Pol I transcription assayed as in a (complementary assay, Supplementary Fig. S6c ). ( d , e ) Inhibition of Top2 or depletion of Top2α reduces the accumulation of PIC components SL1, UBF and Pol I at the rDNA promoter in serum-refed cells. ChIP assays were performed on chromatin from cells treated as in a and c , respectively, using antibodies for Top2α, TAF I 63 (TAF1B; SL1), UBF and A135 (Pol I), and normalized to control IgG samples. s.d. is shown, n =3; *** P

Article Snippet: For Top2 inhibition, Top2 poison etoposide (100 μM final concentration; Merck) or catalytic inhibitors ICRF-193 (50 μM) and merbarone (100 μM; Merck) were added (except ).

Techniques: Activation Assay, Inhibition, Incubation, Chromatin Immunoprecipitation