Silencing Nuclear Pore Protein Tpr Elicits a Senescent-Like Phenotype in Cancer Cells
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Mendeley | Further Information

{"title"=>"Silencing nuclear pore protein Tpr elicits a senescent-like phenotype in cancer cells", "type"=>"journal", "authors"=>[{"first_name"=>"Brigitte", "last_name"=>"David-Watine", "scopus_author_id"=>"6602930269"}], "year"=>2011, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "doi"=>"10.1371/journal.pone.0022423", "pui"=>"362161271", "sgr"=>"79960470360", "pmid"=>"21811608", "scopus"=>"2-s2.0-79960470360"}, "id"=>"8ce3f16a-4b7e-373e-a960-eea7a45616b3", "abstract"=>"BACKGROUND: Tpr is a large coiled-coil protein located in the nuclear basket of the nuclear pore complex for which many different functions were proposed from yeast to human.\\n\\nMETHODOLOGY/PRINCIPAL FINDINGS: Here we show that depletion of Tpr by RNA interference triggers G0-G1 arrest and ultimately induces a senescent-like phenotype dependent on the presence of p53. We also found that Tpr depletion impairs the NES [nuclear export sequence]-dependent nuclear export of proteins and causes partial co-depletion of Nup153. In addition Tpr depletion impacts on level and function of the SUMO-protease SENP2 thus affecting SUMOylation regulation at the nuclear pore and overall SUMOylation in the cell.\\n\\nCONCLUSIONS: Our data for the first time provide evidence that a nuclear pore component plays a role in controlling cellular senescence. Our findings also point to new roles for Tpr in the regulation of SUMO-1 conjugation at the nuclear pore and directly confirm Tpr involvement in the nuclear export of NES-proteins.", "link"=>"http://www.mendeley.com/research/silencing-nuclear-pore-protein-tpr-elicits-senescentlike-phenotype-cancer-cells", "reader_count"=>41, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>1, "Student > Doctoral Student"=>1, "Researcher"=>14, "Student > Ph. D. Student"=>14, "Student > Postgraduate"=>4, "Student > Master"=>1, "Other"=>1, "Student > Bachelor"=>3, "Professor"=>2}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>1, "Student > Doctoral Student"=>1, "Researcher"=>14, "Student > Ph. D. Student"=>14, "Student > Postgraduate"=>4, "Student > Master"=>1, "Other"=>1, "Student > Bachelor"=>3, "Professor"=>2}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>6, "Agricultural and Biological Sciences"=>30, "Medicine and Dentistry"=>2, "Neuroscience"=>1, "Sports and Recreations"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Neuroscience"=>{"Neuroscience"=>1}, "Sports and Recreations"=>{"Sports and Recreations"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>30}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>6}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"Canada"=>2, "Japan"=>1, "United Kingdom"=>1, "Spain"=>1}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/755125"], "description"=>"<p>HeLa cells seeded in 24-well plates at 5 10<sup>−4</sup> cells per well were treated with Tpr or mock siRNAs. 1A. 48 hours after siRNA treatment as indicated on top of the figure, a cell extract was prepared and analyzed by western blotting using anti-Tpr Mab 203-37; tubulin was used as loading control. 1B. Bright field image (right panel) and confocal image (left panel) of HeLa cells labeled with an anti-Tpr monoclonal antibody after 2 days of transfection with Tpr or mock siRNA. Scale bar: 5 µm. 1C. Growth curve of HeLa cells transfected with Tpr, mock siRNA or not transfected: control (Ctrl). 1D. Representative FACS profiles of mock siRNA treated (left panel), Tpr siRNAs treated cells (center) and non-transfected cells or control (right). Ethanol-fixed cells were incubated with PI and analyzed by FACS for ploidy. Cell number is represented on the vertical axis and DNA content on the horizontal axis. G0–G1 and G2-M phases are represented as first and second peaks starting from the vertical axis, respectively. The intermediate striped domain corresponds to the S phase. 1E. Induction of senescence: cells were plated at low density and treated with Tpr or mock siRNAs. After 6 days, cells were fixed and stained for SA-β-gal activity at pH 6.0. Scale bar: 20 µm. 1F. Quantification of SA-β-gal positive cells in cultures depleted of Tpr and stained for SA-β-gal activity at pH 6.0. 1G–1H. U2OS cells were transfected with Tpr or mock siRNAs. 1G: After 6 days the cells were fixed and stained for BrdU incorporation and SA-β-gal activity at pH 6.0. Scale bar: 15 µm. 1H: After 6 days, the cells were labeled with anti-HP1γ and anti-MacroH2A antibodies and DAPI. Scale bar: 5 µm. 1I: A375 cells were transfected with Tpr or mock siRNAs. After 6 days the cells were fixed and stained for SA-β-gal activity at pH 6.0. Scale bar: 15 µm.</p>", "links"=>[], "tags"=>["tpr", "depletion", "hela"], "article_id"=>425504, "categories"=>["Molecular Biology", "Biochemistry", "Cell Biology"], "users"=>["Brigitte David-Watine"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0022423.g001", "stats"=>{"downloads"=>1, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Cell_cycle_effects_of_Tpr_depletion_in_HeLa_cells_/425504", "title"=>"Cell cycle effects of Tpr depletion in HeLa cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-07-19 01:31:44"}
  • {"files"=>["https://ndownloader.figshare.com/files/755673"], "description"=>"<p>After 6 days cells transfected with Tpr or Tpr and SUMO-1, as described in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022423#pone-0022423-g005\" target=\"_blank\">Fig. 5C</a>, were analyzed for SA-β-galactosidase staining. Forty to fifty fields were counted for each condition: the number of fields containing blue cells and the percentage of space occupied in each field by enlarged blue cells were evaluated. SABS: percentage of space occupied by blue cells in a field. Results are given as percentages of fields in the four categories for Tpr or Tpr+SUMO-1 experiments. Numbers between round brackets correspond to standard deviation. (a): only one field with blue cells was observed.</p>", "links"=>[], "tags"=>["sumo-1", "depletion", "tpr-induced"], "article_id"=>426052, "categories"=>["Molecular Biology", "Biochemistry", "Cell Biology"], "users"=>["Brigitte David-Watine"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0022423.t001", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effects_of_SUMO_1_depletion_on_Tpr_induced_senescence_/426052", "title"=>"Effects of SUMO-1 depletion on Tpr-induced senescence.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2011-07-19 01:40:52"}
  • {"files"=>["https://ndownloader.figshare.com/files/755574"], "description"=>"<p>5A–5B. Analysis of the overall level of SUMOylation in cells transfected with Tpr, Nup153, SENP2 and mock siRNAs. 5A. Nuclear extracts of siRNAs treated cells were prepared and analyzed by western blotting using the antibodies as described on the left of the figure. RPB1 was used as loading control. 5B. Analysis of RanGAP1 SUMOylation. Cytoplasmic extracts of Tpr, Nup153, SENP2 and mock siRNAs-treated cells were prepared and analyzed by western blotting using an anti-RanGAP1 antibody recognizing both SUMOylated and non-SUMOylated forms of RanGAP1 and with tubulin as loading control. 5C: SUMO-1 siRNA treatment overrides Tpr depletion-induced senescence in HeLa cells. HeLa cells were transfected with Tpr, SUMO-1, Tpr and SUMO-1 and mock siRNAs. After 6days, the cells were fixed and stained for SA-β-gal activity at pH 6.0. A representative picture of each condition is presented. Scale bar: 15 µm.</p>", "links"=>[], "tags"=>["regulates"], "article_id"=>425957, "categories"=>["Molecular Biology", "Biochemistry", "Cell Biology"], "users"=>["Brigitte David-Watine"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0022423.g005", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Tpr_regulates_SUMOylation_/425957", "title"=>"Tpr regulates SUMOylation.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-07-19 01:39:17"}
  • {"files"=>["https://ndownloader.figshare.com/files/379977", "https://ndownloader.figshare.com/files/380045", "https://ndownloader.figshare.com/files/380089"], "description"=>"<div><h3>Background</h3><p>Tpr is a large coiled-coil protein located in the nuclear basket of the nuclear pore complex for which many different functions were proposed from yeast to human.</p> <h3>Methodology/Principal Findings</h3><p>Here we show that depletion of Tpr by RNA interference triggers G0–G1 arrest and ultimately induces a senescent-like phenotype dependent on the presence of p53. We also found that Tpr depletion impairs the NES [nuclear export sequence]-dependent nuclear export of proteins and causes partial co-depletion of Nup153. In addition Tpr depletion impacts on level and function of the SUMO-protease SENP2 thus affecting SUMOylation regulation at the nuclear pore and overall SUMOylation in the cell.</p> <h3>Conclusions</h3><p>Our data for the first time provide evidence that a nuclear pore component plays a role in controlling cellular senescence. Our findings also point to new roles for Tpr in the regulation of SUMO-1 conjugation at the nuclear pore and directly confirm Tpr involvement in the nuclear export of NES-proteins.</p> </div>", "links"=>[], "tags"=>["pore", "tpr", "elicits", "senescent-like", "phenotype", "cancer", "cells"], "article_id"=>135095, "categories"=>["Molecular Biology", "Biochemistry", "Cell Biology"], "users"=>["Brigitte David-Watine"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0022423.s001", "https://dx.doi.org/10.1371/journal.pone.0022423.s002", "https://dx.doi.org/10.1371/journal.pone.0022423.s003"], "stats"=>{"downloads"=>4, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Silencing_Nuclear_Pore_Protein_Tpr_Elicits_a_Senescent_Like_Phenotype_in_Cancer_Cells/135095", "title"=>"Silencing Nuclear Pore Protein Tpr Elicits a Senescent-Like Phenotype in Cancer Cells", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2011-07-19 01:24:55"}
  • {"files"=>["https://ndownloader.figshare.com/files/755352"], "description"=>"<p>3A–3B. Distribution of GFP in cells co-transfected with STAT1-NES-GFP and Tpr, Crm1 or control (mock) siRNAs. 3A: GFP distribution was analyzed in live cells. Nuclei were labeled with Hoechst 33342. Scale bar: 10 µm. 3B: Cells were then permeabilized and labeled with antibodies specific for Tpr and Crm1 and DAPI for the DNA. Top panel: Tpr depletion and control; lower panel: Crm1 depletion and control. Scale bar: 10 µm. 3C–3D: Tpr depletion delays nuclear export of NFκB. HeLa cells were transfected with Tpr or mock siRNAs 2 days prior to NFκB induction. Transfected and control cells were then incubated with TNF 100 IU/mL for 40 min. TNF was then removed from the medium and the cells were either fixed or kept for another 1h30 in fresh medium before fixation. 3C: Quantification of the NFκB signal: All images were acquired at the same magnification and exposure and the NFκB signal was quantified using the same surface area in the nuclei and cytoplasm of cells under the different experimental conditions using OpenLab3.1.2. The cytosolic signal was plotted against the nuclear signal and standard deviation was calculated using Microsoft Excel. Error bars represent the standard deviation. Note that the cytosolic to nuclear signal ratio is very similar in control cells before TNF treatment and 1h30 after TNF removal. This was as expected and is due to NFκB fully returning to the cytoplasm at this time. 1h30 after TNF removal the cytosolic to nuclear signal ratio was about 25% lower in the Tpr-depleted cells than in the control cells. 3D: Immunofluorescent labeling of NFκB and Tpr 1h30 after TNF removal in Tpr-depleted and control cells. Fixed cells were labeled with a rabbit anti-NFκB antibody and anti-Tpr mAb 203-37.</p>", "links"=>[], "tags"=>["crm1", "nes-proteins", "tpr", "depleted"], "article_id"=>425724, "categories"=>["Molecular Biology", "Biochemistry", "Cell Biology"], "users"=>["Brigitte David-Watine"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0022423.g003", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Analysis_of_Crm1_dependent_NES_proteins_nuclear_export_in_Tpr_depleted_cells_/425724", "title"=>"Analysis of Crm1 dependent NES-proteins nuclear export in Tpr depleted cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-07-19 01:35:24"}
  • {"files"=>["https://ndownloader.figshare.com/files/755460"], "description"=>"<p>4A. HeLa cells were transfected with mock, Tpr or Nup153 siRNAs as indicated on the top. Whole cell lysates were analyzed 2 days post-transfection by immunoblotting using antibodies directed against Tpr, the anti-FG repeat nucleoporin Mab414 to label Nup153 and Nup62 and anti-tubulin as loading control. 4B–4C. Nup153 expression is specifically lowered in Tpr-depleted cells. HeLa cells were treated with Tpr2 or mock siRNAs for 2 days. 4C. Cells were fixed and labeled with an anti-Tpr antibody and anti-emerin, anti-Mab414 or anti-Nup153 antibodies. Scale bar: 15 µm. 4D. Details from Mab414 and Nup153 labeling. Scale bar: 5 µm. 4D. Analysis of SENP2 expression in nuclear extracts of 293 cells treated with Tpr, SENP2 and mock siRNAs by western blot analysis. TRFII is used as a loading control.</p>", "links"=>[], "tags"=>["nup153", "senp2", "proteins", "tpr-depleted"], "article_id"=>425838, "categories"=>["Molecular Biology", "Biochemistry", "Cell Biology"], "users"=>["Brigitte David-Watine"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0022423.g004", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Analysis_of_Nup153_and_SENP2_proteins_in_Tpr_depleted_cells_/425838", "title"=>"Analysis of Nup153 and SENP2 proteins in Tpr-depleted cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-07-19 01:37:18"}
  • {"files"=>["https://ndownloader.figshare.com/files/755248"], "description"=>"<p>2A. Analysis of p53 distribution in HeLa cells by immunofluorescence. Upper panel: HeLa cells were transfected with Tpr, Nup153 and mock siRNAs as indicated on the left; lower panel: HeLa cells were transfected with Nup133 and mock siRNAs. The cells were fixed at day 2 post -transfection and labeled with an anti-Tpr antibody (top left panels) or anti-Nup133 (bottom left panel) and an anti-p53 (right top and bottom panels). 2B. Hela cell whole cell protein lysates were separated by SDS-PAGE and immunoblotted with antibodies specific for Tpr, p53, p21, p16 and tubulin as indicated in the <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022423#pone-0022423-g002\" target=\"_blank\">figure. 2C</a>. Whole cell protein lysates of U2OS cells treated with Tpr, Tpr+p53 or control (mock) siRNAs were separated by SDS-PAGE and immunoblotted with antibodies specific for Tpr, p53, p21 and tubulin as indicated in the <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022423#pone-0022423-g002\" target=\"_blank\">figure. 2D</a>. Co-depletion of p53 and Tpr reversed senescence induction. Cells were transfected with mock, Tpr, p53 or Tpr and p53 in combinations as indicated. The final siRNA concentration was 100 nM in each case and compensation was made with mock siRNAs. Cells were labeled for SA-β-galactosidase at 6 days post-transfection and the result is given as the percentage of SA-β-galactosidase positive cells in each of the three independent experiments.</p>", "links"=>[], "tags"=>["stabilized", "accumulates", "nuclei", "tpr-depleted"], "article_id"=>425621, "categories"=>["Molecular Biology", "Biochemistry", "Cell Biology"], "users"=>["Brigitte David-Watine"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0022423.g002", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_p53_is_stabilized_and_accumulates_in_the_nuclei_of_Tpr_depleted_cells_/425621", "title"=>"p53 is stabilized and accumulates in the nuclei of Tpr-depleted cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-07-19 01:33:41"}

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Relative Metric

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