Journal:

Article Title: Poisoning of human DNA topoisomerase I by ecteinascidin 743, an anticancer drug that selectively alkylates DNA in the minor groove

doi:

Figure Lengend Snippet: Et743 induces top1-mediated DNA single-strand breaks. SV40 DNA was reacted with top1 in the absence or presence of drugs, as indicated. Reactions were stopped with 0.5% SDS followed by proteinase K incubation. Samples were run in 1% agarose gels containing ethidium bromide. Percent nicked DNA was computed after FluorImager analysis (Molecular Dynamics) by using the imagequant software and is indicated below each lane. (A) top1-mediated nicking assay by using supercoiled negative SV40 DNA. Lane 1, DNA alone; lane 2, + top1; and lanes 3–6, + top1 and the indicated concentrations of Et743 or CPT. (B) Et743 induces more top1-mediated DNA nicking in supercoiled than in linear SV40 DNA. Lanes 1 and 5, DNA alone; lanes 2 and 6, DNA + top1; lanes 3 and 7, DNA + top1 + Et743 (0.1 μM); lanes 4 and 8, Et743 without top1. (C) Et743 does not induce detectable top2 cleavage complexes. Lane 1, DNA alone; lane 2, + top2; and lanes 3–4, + top2 and the indicated drug concentrations.

Article Snippet: Each reaction (10 μl final volume) in 10 mM Tris⋅HCl, pH 7.5/50 mM KCl/5 mM MgCl 2 /0.1 mM EDTA/1 mM ATP/15 μg/ml BSA contained 0.3 μg supercoiled simian virus 40 (SV40) DNA (GIBCO/BRL).

Techniques: Incubation, Software

Journal:

Article Title: Poisoning of human DNA topoisomerase I by ecteinascidin 743, an anticancer drug that selectively alkylates DNA in the minor groove

doi:

Figure Lengend Snippet: DNA alkylation by Et743 induces salt-reversible top1-mediated DNA breaks at different sites from CPT. (A) Differences in sequence selectivity of top1-mediated DNA breaks for Et743 and CPT. The long BanI-HpaII fragments of SV40 DNA 3′-end-labeled at the BanI Site was used. Lane 1, formic acid (FA); lane 2, DNA alone; lane 3, + top1 without drug; lane 4, top1 + Et743 (10 μM); lane 5, top1 + CPT (10 μM). (B) Salt reversibility of the top1-mediated DNA breaks induced by Et743. Drug-induced DNA cleavage was reversed by adding 0.35 M NaCl (final concentration) (at time 0 corresponding to a 30-min incubation of top1 with Et743 or CPT; lanes 3 and 8, respectively) and by further incubation at 25°C for the indicated times. (C) Et743-DNA adducts induce the top1 cleavage complexes. 3′-end-labeled PvuII/HindIII fragment of pSK(−) phagemid DNA was used for these reactions. In lanes 2–9, reactions were performed in two consecutive steps. First, the DNA was treated overnight (O/N ≈16 hr) with or without drug, as indicated above pairs of lanes, then DNA was ethanol precipitated to remove free drug. Secondly, the DNA was reacted ± top1 in the absence of added drug (lanes 2–10) or in the presence of 10 μM CPT (lane 11). Lane 1, FA-sequencing lane.

Article Snippet: Each reaction (10 μl final volume) in 10 mM Tris⋅HCl, pH 7.5/50 mM KCl/5 mM MgCl 2 /0.1 mM EDTA/1 mM ATP/15 μg/ml BSA contained 0.3 μg supercoiled simian virus 40 (SV40) DNA (GIBCO/BRL).

Techniques: Sequencing, Labeling, Concentration Assay, Incubation

Journal: PLoS Pathogens

Article Title: Nuclear Export and Import of Human Hepatitis B Virus Capsid Protein and Particles

doi: 10.1371/journal.ppat.1001162

Figure Lengend Snippet: The definitions of the patterns “N>C”, “C>N”, and “C+N” are as described in the legend to . Fig. 3B . Immunofluorescence assay of wild type and various chimeric SV40 LT fused in-frame with wild type or mutant HBc ARD, as illustrated in Fig. 3A. Approximately 300 to 500 cells for each construct were scored for their subcellular localization of SV40 LT. SV40 LT (green), lamin B1 (red), and DAPI (blue). The control sample is from mock transfected Huh7 cells. Fig. 3C . Western blot analysis of various chimeric SV40 LT is illustrated in Fig. 3A. Fig. 3D–3F : Both HBc ARD-I and ARD-III are required for nuclear localization of NLS-deficient SV40 LT (mutant K128T). When the NLS of SV40 LT was abolished by mutation K128T, 100% of cells displayed cytoplasmic LT (C>N pattern). In contrast, approximately 54% of cells transfected with plasmid SV40LT (K128T)-HBc ARD-II+IV shifted from cytoplasm to nucleus (12% N>C+42% C+N pattern). Fig. 3E . Immunofluorescence assay of various chimeric SV40 LT (NLS-defective) as illustrated in Fig. 3D. Fig. 3F . Western blot analysis of various chimeric SV40 LT (NLS-defective) as illustrated in Fig. 3D. The weak bandings of SV40LT at lanes b, c, and d in the nuclear fraction are likely from the residual contamination from the cytoplasmic fraction.

Article Snippet: Other antibodies used in this paper include mouse monoclonal anti-HBc antibody (Hyb-3120, Institute of Immunology, Japan) , , , mouse monoclonal anti-alpha-tubulin antibody (ICON-GeneTex, Taiwan), rabbit polyclonal anti-lamin B1 antibody (ICON-GeneTex, Taiwan), chicken polyclonal anti-Rev (HIV-1) antibody (ICON-GeneTex, Taiwan), mouse and rabbit anti-TAP antibodies (Santa Cruz and ICON-GeneTex, Taiwan), and mouse monoclonal anti-SV40 LT antibody (Abcam).

Techniques: Immunofluorescence, Mutagenesis, Construct, Control, Transfection, Western Blot, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: Nuclear Export and Import of Human Hepatitis B Virus Capsid Protein and Particles

doi: 10.1371/journal.ppat.1001162

Figure Lengend Snippet: Upper panel , Human Huh7 cells were transfected with pCMV-Rev (green) before fusion with Huh7 cells expressing SV40 LT (red). Lower panel , Human Huh7 cells were transfected with pCMV-Rev-HBc ARD (green) before fusion with Huh7 cells expressing SV40 LT (red). DAPI (blue). No nuclear Rev signal was detected.

Article Snippet: Other antibodies used in this paper include mouse monoclonal anti-HBc antibody (Hyb-3120, Institute of Immunology, Japan) , , , mouse monoclonal anti-alpha-tubulin antibody (ICON-GeneTex, Taiwan), rabbit polyclonal anti-lamin B1 antibody (ICON-GeneTex, Taiwan), chicken polyclonal anti-Rev (HIV-1) antibody (ICON-GeneTex, Taiwan), mouse and rabbit anti-TAP antibodies (Santa Cruz and ICON-GeneTex, Taiwan), and mouse monoclonal anti-SV40 LT antibody (Abcam).

Techniques: Transfection, Expressing

Journal: PLoS Pathogens

Article Title: Nuclear Export and Import of Human Hepatitis B Virus Capsid Protein and Particles

doi: 10.1371/journal.ppat.1001162

Figure Lengend Snippet: Huh7 donor cells transfected with various versions of SV40LT-HBc chimera (green) were fused with Huh7 recipient cells transfected with NES-deficient Rev (red). DAPI (blue). a) SV40 large T antigen is localized exclusively to the nucleus. There was no transport of SV40 LT from donor to recipient nuclei. b) Chimeric protein of SV40 LT-HBc appeared to shuttle from donor to recipient nuclei. c) Mutant HBc ARD-I+III+IV contains an intact subdomain ARD-II. When this mutant ARD-I+III+IV was fused with SV40 LT, shuttling appeared to occur via the intact ARD-II. d) Mutant HBc ARD-I+II+III contains an intact subdomain ARD-IV. When this mutant ARD-I+II+III was fused with SV40 LT, shuttling appeared to occur via the intact ARD-IV. e) No apparent shuttling was observed for mutant ARD-II+IV, when both ARD-II and ARD-IV subdomains were inactivated. This consistent negative result was based on the examination of a total of 50–60 homokaryons in each experiment, and was controlled by the positive results in panels 5b, 5c, and 5d. Similar results were obtained using a heterokaryon analysis (Supporting Information ).

Article Snippet: Other antibodies used in this paper include mouse monoclonal anti-HBc antibody (Hyb-3120, Institute of Immunology, Japan) , , , mouse monoclonal anti-alpha-tubulin antibody (ICON-GeneTex, Taiwan), rabbit polyclonal anti-lamin B1 antibody (ICON-GeneTex, Taiwan), chicken polyclonal anti-Rev (HIV-1) antibody (ICON-GeneTex, Taiwan), mouse and rabbit anti-TAP antibodies (Santa Cruz and ICON-GeneTex, Taiwan), and mouse monoclonal anti-SV40 LT antibody (Abcam).

Techniques: Transfection, Mutagenesis

Journal: PLoS Pathogens

Article Title: Nuclear Export and Import of Human Hepatitis B Virus Capsid Protein and Particles

doi: 10.1371/journal.ppat.1001162

Figure Lengend Snippet: Immunofluorescence assay of Rev or SV40 LT-HBc ARD signals was performed using transfected Huh7 cells with or without leptomycin B treatment. Rev and SV40 LT (green) and DAPI (blue). a) Wild type Rev in Huh7 cells exhibited both nuclear and cytoplasmic Rev (left panel, mock). Upon treatment with leptomycin B, cytoplasmic Rev was lost (right panel). b) When SV40 LT was fused with wild type HBc ARD, SV40 LT was distributed in both cytoplasm and nucleus in approximately 50% of transfected Huh7 cells . The percentage of cells exhibiting this C+N pattern of SV40 LT was not affected upon treatment with leptomycin B for 12 hrs (data not shown).

Article Snippet: Other antibodies used in this paper include mouse monoclonal anti-HBc antibody (Hyb-3120, Institute of Immunology, Japan) , , , mouse monoclonal anti-alpha-tubulin antibody (ICON-GeneTex, Taiwan), rabbit polyclonal anti-lamin B1 antibody (ICON-GeneTex, Taiwan), chicken polyclonal anti-Rev (HIV-1) antibody (ICON-GeneTex, Taiwan), mouse and rabbit anti-TAP antibodies (Santa Cruz and ICON-GeneTex, Taiwan), and mouse monoclonal anti-SV40 LT antibody (Abcam).

Techniques: Immunofluorescence, Transfection

Journal: PLoS Pathogens

Article Title: Nuclear Export and Import of Human Hepatitis B Virus Capsid Protein and Particles

doi: 10.1371/journal.ppat.1001162

Figure Lengend Snippet: Fig. 7A . Huh7 cells were transfected with expression vectors of a) SV40 LT, or b) SV40 LT-HBc ARD chimera. Transfected cell lysates were immunoprecipitated (IP) with anti-SV40 LT antibody. Equal amounts of immunoprecipitated cell lysates from a) or b) were loaded on SDS-PAGE, respectively, followed by Western blot analysis using anti-TAP or anti-SV40 LT antibodies. The 66 kDa TAP protein can be detected only in cell lysates transfected with SV40 LT-HBc ARD, but not transfected with wild type SV40 LT, indicating that TAP could bind to HBc ARD. This result was confirmed in a reciprocal experiment using an anti-TAP antibody for IP. Fig. 7B . Knockdown of endogenous TAP protein by siRNA against TAP ( panel b ) resulted in a more than 17-fold increase of nuclear accumulation of wild type HBc (N>C pattern shifted from 3% to 52%). In contrast, control siRNA (Nontarget) ( panel a ) did not affect the subcellular localization pattern of HBc. HBc (red), α-tubulin (green) and DAPI (blue). Fig. 7C . Treatment of Huh7 cells with TAP-specific siRNA resulted in appreciable reduction of TAP protein by Western blot analysis using anti-TAP antibody. * Non-specific bands served as an internal control. (cf for detail). Fig. 7D . The nuclear predominant phenotype of wild type HBc in Huh7 cells treated with siTAP can be reverted efficiently to a cytoplasmic predominant phenotype by cotransfection with a CMV-TAP expression vector. Fig. 7E . Western blot analysis of TAP protein in Huh7 cells transfected with a vector only or a plasmid CMV-TAP. Fig. 7F . Upon treatment with siTAP, wild type HBV DNA synthesis in Huh7 cells was reduced approximately 7-fold by Southern blot analysis. RC: relaxed circle DNA, SS: single-strand DNA. While there was no difference in the results of MTT cytotoxicity assay using media from cell culture with or without siTAP treatment, the HBsAg level by ELISA was reduced by approximately 10-fold with siTAP treatment. The averaged values of DNA intensity, MTT assay, and HBsAg assay were processed from four independent experiments .

Article Snippet: Other antibodies used in this paper include mouse monoclonal anti-HBc antibody (Hyb-3120, Institute of Immunology, Japan) , , , mouse monoclonal anti-alpha-tubulin antibody (ICON-GeneTex, Taiwan), rabbit polyclonal anti-lamin B1 antibody (ICON-GeneTex, Taiwan), chicken polyclonal anti-Rev (HIV-1) antibody (ICON-GeneTex, Taiwan), mouse and rabbit anti-TAP antibodies (Santa Cruz and ICON-GeneTex, Taiwan), and mouse monoclonal anti-SV40 LT antibody (Abcam).

Techniques: Transfection, Expressing, Immunoprecipitation, SDS Page, Western Blot, Knockdown, Control, Cotransfection, Plasmid Preparation, DNA Synthesis, Southern Blot, Cytotoxicity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, MTT Assay, HBsAg Assay