Quantitative Live Imaging of Endogenous DNA Replication in Mammalian Cells
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{"title"=>"Quantitative Live Imaging of Endogenous DNA Replication in Mammalian Cells", "type"=>"journal", "authors"=>[{"first_name"=>"Andrew", "last_name"=>"Burgess", "scopus_author_id"=>"7201495301"}, {"first_name"=>"Thierry", "last_name"=>"Lorca", "scopus_author_id"=>"55893817800"}, {"first_name"=>"Anna", "last_name"=>"Castro", "scopus_author_id"=>"25947329700"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"pui"=>"365718541", "sgr"=>"84866702125", "issn"=>"19326203", "pmid"=>"23029203", "scopus"=>"2-s2.0-84866702125", "doi"=>"10.1371/journal.pone.0045726", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)"}, "id"=>"08744db9-b88d-3cea-8672-ffea477f351d", "abstract"=>"Historically, the analysis of DNA replication in mammalian tissue culture cells has been limited to static time points, and the use of nucleoside analogues to pulse-label replicating DNA. Here we characterize for the first time a novel Chromobody cell line that specifically labels endogenous PCNA. By combining this with high-resolution confocal time-lapse microscopy, and with a simplified analysis workflow, we were able to produce highly detailed, reproducible, quantitative 4D data on endogenous DNA replication. The increased resolution allowed accurate classification and segregation of S phase into early-, mid-, and late-stages based on the unique subcellular localization of endogenous PCNA. Surprisingly, this localization was slightly but significantly different from previous studies, which utilized over-expressed GFP tagged forms of PCNA. Finally, low dose exposure to Hydroxyurea caused the loss of mid- and late-S phase localization patterns of endogenous PCNA, despite cells eventually completing S phase. Taken together, these results indicate that this simplified method can be used to accurately identify and quantify DNA replication under multiple and various experimental conditions.", "link"=>"http://www.mendeley.com/research/quantitative-live-imaging-endogenous-dna-replication-mammalian-cells", "reader_count"=>69, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>5, "Researcher"=>14, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>25, "Student > Master"=>10, "Student > Bachelor"=>10, "Lecturer > Senior Lecturer"=>1, "Professor"=>3}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>5, "Researcher"=>14, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>25, "Student > Master"=>10, "Student > Bachelor"=>10, "Lecturer > Senior Lecturer"=>1, "Professor"=>3}, "reader_count_by_subject_area"=>{"Engineering"=>1, "Biochemistry, Genetics and Molecular Biology"=>14, "Agricultural and Biological Sciences"=>50, "Medicine and Dentistry"=>2, "Computer Science"=>1, "Immunology and Microbiology"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>50}, "Computer Science"=>{"Computer Science"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>14}}, "reader_count_by_country"=>{"United States"=>1, "Poland"=>1, "France"=>1, "Germany"=>1}, "group_count"=>2}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/573981"], "description"=>"<p>(<b>A</b>) HeLa Chromobody (Chromotek) cells were blocked in G1/S with thymidine for 24 h. Cells were released and then pulsed for 20 min with 10 µM EdU (Invitrogen) at 4 h and 6 h post release to label mid- and late- replicating cells. Cells were fixed and stained using the EdU Click-iT labeling kit as per the manufactures instructions (Invitrogen). Shown are the maximum image projections from 0.3 µm serial Z-sections. High levels of co-localization (Yellow) are clearly observed in the overlaid images between the PCNA targeting Chromobody (Green) and EdU incorporation (Red), Scale bare equals 5 µm. (<b>B</b>) Schematic workflow showing the simplified method used in this study to capture, process and analyze data. (<b>C</b>) HeLa Chromobody cells (Chromotek), were synchronized in G1/S and analyzed by 4D microscopy (3D + time). Duplicate samples were taken every 2 h and processed by flow cytometry to confirm that the majority of cells were progressing through S phase. Shown are the maximum projection images (Top) from a typical cell as it progresses through S phase. Dotted lines indicate the position of the Z-section slice taken for the side- view (Side) yellow arrows indicate the nucleoli. The results from the automated dot-tracking feature from Imaris software (Bitplane Inc.) are also shown (Overlay). Scale bar 5 µm.</p>", "links"=>[], "tags"=>["chromobody", "dna", "replication"], "article_id"=>244480, "categories"=>["Physics", "Molecular Biology", "Cell Biology", "Biophysics"], "users"=>["Andrew Burgess", "Thierry Lorca", "Anna Castro"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0045726.g001", "stats"=>{"downloads"=>1, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Using_a_novel_Chromobody_cell_line_to_image_DNA_replication_in_live_cells_/244480", "title"=>"Using a novel Chromobody cell line to image DNA replication in live cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-09-20 01:14:40"}
  • {"files"=>["https://ndownloader.figshare.com/files/574193"], "description"=>"<p>Maximum image projection (MIP), and automated dot tracking overlay (Overlay) images from a mid- (purple) and late- S (green) phase cell, and the corresponding zoom cropped sections highlighting the nucleoli and peripheral foci respectively. Horizontal segregation of the nucleolus (yellow dotted line) clearly shows the top half undergoing replication at earlier time points, before shifting to the lower half. However, over time no significant movement of individual dots was observed in either mid- or late- locations (yellow arrows), orange arrows indicates point where a focus is resolved. Scale bar 1 µm, time expressed as hh:mm.</p>", "links"=>[], "tags"=>["dots", "moving", "replication"], "article_id"=>244688, "categories"=>["Physics", "Molecular Biology", "Cell Biology", "Biophysics"], "users"=>["Andrew Burgess", "Thierry Lorca", "Anna Castro"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0045726.g003", "stats"=>{"downloads"=>1, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Individual_dots_resolve_before_moving_to_other_replication_sites_/244688", "title"=>"Individual dots resolve before moving to other replication sites.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-09-20 01:18:08"}
  • {"files"=>["https://ndownloader.figshare.com/files/302972", "https://ndownloader.figshare.com/files/302988", "https://ndownloader.figshare.com/files/303009", "https://ndownloader.figshare.com/files/303045", "https://ndownloader.figshare.com/files/303082", "https://ndownloader.figshare.com/files/303110", "https://ndownloader.figshare.com/files/303210", "https://ndownloader.figshare.com/files/303234"], "description"=>"<div><p>Historically, the analysis of DNA replication in mammalian tissue culture cells has been limited to static time points, and the use of nucleoside analogues to pulse-label replicating DNA. Here we characterize for the first time a novel Chromobody cell line that specifically labels endogenous PCNA. By combining this with high-resolution confocal time-lapse microscopy, and with a simplified analysis workflow, we were able to produce highly detailed, reproducible, quantitative 4D data on endogenous DNA replication. The increased resolution allowed accurate classification and segregation of S phase into early-, mid-, and late-stages based on the unique subcellular localization of endogenous PCNA. Surprisingly, this localization was slightly but significantly different from previous studies, which utilized over-expressed GFP tagged forms of PCNA. Finally, low dose exposure to Hydroxyurea caused the loss of mid- and late-S phase localization patterns of endogenous PCNA, despite cells eventually completing S phase. Taken together, these results indicate that this simplified method can be used to accurately identify and quantify DNA replication under multiple and various experimental conditions.</p> </div>", "links"=>[], "tags"=>["quantitative", "imaging", "endogenous", "dna", "replication", "mammalian", "cells"], "article_id"=>119750, "categories"=>["Physics", "Molecular Biology", "Cell Biology", "Biophysics"], "users"=>["Andrew Burgess", "Thierry Lorca", "Anna Castro"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0045726.s001", "https://dx.doi.org/10.1371/journal.pone.0045726.s002", "https://dx.doi.org/10.1371/journal.pone.0045726.s003", "https://dx.doi.org/10.1371/journal.pone.0045726.s004", "https://dx.doi.org/10.1371/journal.pone.0045726.s005", "https://dx.doi.org/10.1371/journal.pone.0045726.s006", "https://dx.doi.org/10.1371/journal.pone.0045726.s007", "https://dx.doi.org/10.1371/journal.pone.0045726.s008"], "stats"=>{"downloads"=>11, "page_views"=>41, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Quantitative_Live_Imaging_of_Endogenous_DNA_Replication_in_Mammalian_Cells/119750", "title"=>"Quantitative Live Imaging of Endogenous DNA Replication in Mammalian Cells", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2012-09-20 02:42:30"}
  • {"files"=>["https://ndownloader.figshare.com/files/574331"], "description"=>"<p>(<b>A</b>) DNA content FACS analysis of thymidine synchronized HeLa Chromobody cells treated with 200 µM of HU at time of release. Cells were harvested at 0, 8, 12, 16, and 20 hours after release from G1/S. Data clearly shows that HU treated cells eventually complete S phase and transit through mitosis and back into G1 phase after 16–20 hours. (<b>B</b>) Shown are the maximum image projections (MIP) and the corresponding overlaid Imaris dot classification in green (Overlay) from time-lapse movies of HeLa Chromobody cells synchronized and treated with low dose Hydroxyurea (200 µm). Time is expressed in hh:mm, scale bar is 2 µm. Arrows highlight examples of nucleoli (orange) and peripheral (yellow) foci localizations. (<b>C</b>) As per <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045726#pone-0045726-g002\" target=\"_blank\">Figure 2A</a>, except as cells were released from G1/S they were treated with low dose Hydroxyurea (200 µM). Shown is a representative graph from a single cell over time. (<b>D</b>) A compilation bar graph detailing the fate of 15 individual HU treated cells.</p>", "links"=>[], "tags"=>["hu", "blocks", "mid", "origins"], "article_id"=>244825, "categories"=>["Physics", "Molecular Biology", "Cell Biology", "Biophysics"], "users"=>["Andrew Burgess", "Thierry Lorca", "Anna Castro"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0045726.g004", "stats"=>{"downloads"=>1, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Low_dose_HU_blocks_mid_and_late_origins_of_replication_/244825", "title"=>"Low dose HU blocks mid and late origins of replication.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-09-20 01:20:25"}
  • {"files"=>["https://ndownloader.figshare.com/files/574081"], "description"=>"<p>(<b>A</b>) Dot quantification data was exported from Imaris, and compiled into graphs. The total number of dots was plotted and overlaid with the maximum foci volume at each time point. Cells were separated into G1, early-, mid-, late- S phase, G2 and Mitosis (M) as per methods, shown is a representative graph from a single cell over time. Inset shows typical maximum projection images of late- S, G2 and early mitotic cells. Dotted white line indicates location of the nuclear envelope before and after breakdown (NEBD). (<b>B</b>) A compilation bar graph detailing the high level of reproducibility in timing of each cell cycle phase (G1, early-, mid-, late-, G2, Mitosis) for 15 individual control cells.</p>", "links"=>[], "tags"=>["imaging"], "article_id"=>244586, "categories"=>["Physics", "Molecular Biology", "Cell Biology", "Biophysics"], "users"=>["Andrew Burgess", "Thierry Lorca", "Anna Castro"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0045726.g002", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Quantification_of_Live_Imaging_Data_/244586", "title"=>"Quantification of Live Imaging Data.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-09-20 01:16:26"}

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  • {"unique-ip"=>"19", "full-text"=>"18", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"9"}
  • {"unique-ip"=>"17", "full-text"=>"19", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"4", "cited-by"=>"0", "year"=>"2019", "month"=>"10"}

Relative Metric

{"start_date"=>"2012-01-01T00:00:00Z", "end_date"=>"2012-12-31T00:00:00Z", "subject_areas"=>[{"subject_area"=>"/Biology and life sciences/Biochemistry", "average_usage"=>[316, 541, 663, 766, 856, 950, 1041, 1128, 1218, 1302, 1382, 1456, 1526, 1593, 1657, 1729, 1796, 1862, 1930, 1999, 2065, 2132, 2202, 2261, 2319]}, {"subject_area"=>"/Physical sciences/Chemistry", "average_usage"=>[302, 508, 622, 720, 804, 888, 973, 1054, 1141, 1219, 1299, 1370, 1442, 1511, 1574, 1644, 1711, 1782, 1846, 1911, 1971, 2030, 2097, 2155, 2217]}]}
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