Zinc Finger Nuclease Mediated Knockout of ADP-Dependent Glucokinase in Cancer Cell Lines: Effects on Cell Survival and Mitochondrial Oxidative Metabolism
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{"title"=>"Zinc Finger Nuclease Mediated Knockout of ADP-Dependent Glucokinase in Cancer Cell Lines: Effects on Cell Survival and Mitochondrial Oxidative Metabolism", "type"=>"journal", "authors"=>[{"first_name"=>"Susan", "last_name"=>"Richter", "scopus_author_id"=>"57198118095"}, {"first_name"=>"Shona", "last_name"=>"Morrison", "scopus_author_id"=>"36082989100"}, {"first_name"=>"Tim", "last_name"=>"Connor", "scopus_author_id"=>"26649796200"}, {"first_name"=>"Jiechuang", "last_name"=>"Su", "scopus_author_id"=>"54793653300"}, {"first_name"=>"Cristin G.", "last_name"=>"Print", "scopus_author_id"=>"6603682808"}, {"first_name"=>"Ron S.", "last_name"=>"Ronimus", "scopus_author_id"=>"6701842431"}, {"first_name"=>"Sean L.", "last_name"=>"McGee", "scopus_author_id"=>"7004322315"}, {"first_name"=>"William R.", "last_name"=>"Wilson", "scopus_author_id"=>"7404032534"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"pmid"=>"23799003", "issn"=>"19326203", "doi"=>"10.1371/journal.pone.0065267", "pui"=>"369122566", "isbn"=>"1932-6203 (Electronic)\\n1932-6203 (Linking)", "scopus"=>"2-s2.0-84879047939", "sgr"=>"84879047939"}, "id"=>"5010b59c-5068-37d9-a638-3cd6949334b0", "abstract"=>"Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of ADPGK, which encodes an ADP-dependent glucokinase (ADPGK), in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116). All four clones had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002) and 4.3±0.8% (p = 0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis in these lines.", "link"=>"http://www.mendeley.com/research/zinc-finger-nuclease-mediated-knockout-adpdependent-glucokinase-cancer-cell-lines-effects-cell-survi", "reader_count"=>30, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>2, "Student > Doctoral Student"=>4, "Researcher"=>10, "Student > Ph. D. Student"=>8, "Student > Master"=>3, "Other"=>1, "Student > Bachelor"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>2, "Student > Doctoral Student"=>4, "Researcher"=>10, "Student > Ph. D. Student"=>8, "Student > Master"=>3, "Other"=>1, "Student > Bachelor"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>2, "Environmental Science"=>1, "Biochemistry, Genetics and Molecular Biology"=>5, "Medicine and Dentistry"=>5, "Agricultural and Biological Sciences"=>15, "Neuroscience"=>1, "Chemistry"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>5}, "Neuroscience"=>{"Neuroscience"=>1}, "Chemistry"=>{"Chemistry"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>15}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>5}, "Unspecified"=>{"Unspecified"=>2}, "Environmental Science"=>{"Environmental Science"=>1}}, "reader_count_by_country"=>{"United States"=>1, "Spain"=>1}, "group_count"=>0}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1088627"], "description"=>"<p>(A/B). Cell density after seeding 24-well plates at 10<sup>5</sup>/ml. Values are mean and SEM for triplicate cultures. (C/D). Phase contrast microscopy (10× objective magnification) of WT and knockout clones in T flasks.</p>", "links"=>[], "tags"=>["Biochemistry", "Bioenergetics", "Energy-producing organelles", "enzymes", "Enzyme metabolism", "metabolism", "Oxygen metabolism", "Computational biology", "microarrays", "genetics", "gene expression", "Gene function", "Genetic mutation", "immunology", "Immunologic techniques", "Immunohistochemical analysis", "Model organisms", "Animal models", "mouse", "Molecular cell biology", "RNA interference", "Cell growth", "statistics", "Biostatistics", "oncology", "Basic cancer research", "knockout", "clones", "hct116", "h460", "morphology", "respective", "wt", "lines"], "article_id"=>721421, "categories"=>["Biological Sciences", "Medicine", "Mathematics"], "users"=>["Susan Richter", "Shona Morrison", "Tim Connor", "Jiechuang Su", "Cristin G. Print", "Ron S. Ronimus", "Sean L. McGee", "William R. Wilson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0065267.g003", "stats"=>{"downloads"=>3, "page_views"=>126, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_i_ADPGK_i_knockout_clones_of_HCT116_A_C_and_H460_B_D_show_growth_and_morphology_similar_to_the_respective_WT_lines_i_in_vitro_i_/721421", "title"=>"<i>ADPGK</i> knockout clones of HCT116 (A, C) and H460 (B, D) show growth and morphology similar to the respective WT lines <i>in vitro</i>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-06-14 00:23:41"}
  • {"files"=>["https://ndownloader.figshare.com/files/1088640", "https://ndownloader.figshare.com/files/1088674", "https://ndownloader.figshare.com/files/1088672", "https://ndownloader.figshare.com/files/1088669", "https://ndownloader.figshare.com/files/1088661", "https://ndownloader.figshare.com/files/1088657", "https://ndownloader.figshare.com/files/1088653", "https://ndownloader.figshare.com/files/1088650", "https://ndownloader.figshare.com/files/1088648", "https://ndownloader.figshare.com/files/1088647", "https://ndownloader.figshare.com/files/1088646", "https://ndownloader.figshare.com/files/1088645", "https://ndownloader.figshare.com/files/1088644", "https://ndownloader.figshare.com/files/1088642", "https://ndownloader.figshare.com/files/1088641", "https://ndownloader.figshare.com/files/1088678"], "description"=>"<div><p>Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of <i>ADPGK</i>, which encodes an ADP-dependent glucokinase (ADPGK), in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116). All four clones had frameshift mutations in all alleles at the target site in exon 1 of <i>ADPGK,</i> and were ADPGK-null by immunoblotting. <i>ADPGK</i> knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002) and 4.3±0.8% (p = 0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when <i>ADPGK</i> was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of <i>ADPGK</i> in HCT116 cells caused few changes in global gene expression, knockout of <i>ADPGK</i> in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the <i>ADPGK</i> knockouts, in some cases markedly so. Collectively, the results demonstrate that <i>ADPGK</i> can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of <i>ADPGK</i> is cell line dependent and appears to be unrelated to priming of glycolysis in these lines.</p></div>", "links"=>[], "tags"=>["Biochemistry", "Bioenergetics", "Energy-producing organelles", "enzymes", "Enzyme metabolism", "metabolism", "Oxygen metabolism", "Computational biology", "microarrays", "genetics", "gene expression", "Gene function", "Genetic mutation", "immunology", "Immunologic techniques", "Immunohistochemical analysis", "Model organisms", "Animal models", "mouse", "Molecular cell biology", "RNA interference", "Cell growth", "statistics", "Biostatistics", "oncology", "Basic cancer research", "nuclease", "mediated", "knockout", "adp-dependent", "glucokinase", "cancer", "mitochondrial", "oxidative"], "article_id"=>721434, "categories"=>["Biological Sciences", "Medicine", "Mathematics"], "users"=>["Susan Richter", "Shona Morrison", "Tim Connor", "Jiechuang Su", "Cristin G. Print", "Ron S. Ronimus", "Sean L. McGee", "William R. Wilson"], "doi"=>[nil, nil, nil, nil, nil, nil, nil, nil, nil, nil, nil, nil, nil, nil, nil, nil], "stats"=>{"downloads"=>32, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Zinc_Finger_Nuclease_Mediated_Knockout_of_ADP_Dependent_Glucokinase_in_Cancer_Cell_Lines_Effects_on_Cell_Survival_and_Mitochondrial_Oxidative_Metabolism/721434", "title"=>"Zinc Finger Nuclease Mediated Knockout of ADP-Dependent Glucokinase in Cancer Cell Lines: Effects on Cell Survival and Mitochondrial Oxidative Metabolism", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-06-14 00:23:54"}
  • {"files"=>["https://ndownloader.figshare.com/files/1088635"], "description"=>"<p>Kaplan-Meier plots showing time for HCT116 (A) and H460 (B) tumours to reach endpoint. 5-6 mice/group. (C) Immunoblots of lysates from fresh frozen tumours and mouse liver at the tumour growth endpoint. (D) Proportion of tumour area which is necrotic, as determined by H&amp;E staining of histological sections at endpoint (4 tumours/group). (E) Proportion of tumour sections positive for the hypoxia probe pimonidazole, by immunostaining, in the same tumours as for D.</p>", "links"=>[], "tags"=>["Biochemistry", "Bioenergetics", "Energy-producing organelles", "enzymes", "Enzyme metabolism", "metabolism", "Oxygen metabolism", "Computational biology", "microarrays", "genetics", "gene expression", "Gene function", "Genetic mutation", "immunology", "Immunologic techniques", "Immunohistochemical analysis", "Model organisms", "Animal models", "mouse", "Molecular cell biology", "RNA interference", "Cell growth", "statistics", "Biostatistics", "oncology", "Basic cancer research", "xenograft", "wt", "knockout"], "article_id"=>721429, "categories"=>["Biological Sciences", "Medicine", "Mathematics"], "users"=>["Susan Richter", "Shona Morrison", "Tim Connor", "Jiechuang Su", "Cristin G. Print", "Ron S. Ronimus", "Sean L. McGee", "William R. Wilson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0065267.g010", "stats"=>{"downloads"=>0, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Tumour_xenograft_growth_from_i_ADPGK_i_WT_and_knockout_cell_lines_/721429", "title"=>"Tumour xenograft growth from <i>ADPGK</i> WT and knockout cell lines.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-06-14 00:23:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/1088634"], "description"=>"<p><i>PPARGC1A</i> expression was measured by qPCR in WT and the two <i>ADPGK-</i>null clones, and in H460 WT cells two days after treatment with control or <i>ADPGK</i> siRNA. Standard errors are shown for 3 replicate cultures with 3 technical replicates each.</p>", "links"=>[], "tags"=>["Biochemistry", "Bioenergetics", "Energy-producing organelles", "enzymes", "Enzyme metabolism", "metabolism", "Oxygen metabolism", "Computational biology", "microarrays", "genetics", "gene expression", "Gene function", "Genetic mutation", "immunology", "Immunologic techniques", "Immunohistochemical analysis", "Model organisms", "Animal models", "mouse", "Molecular cell biology", "RNA interference", "Cell growth", "statistics", "Biostatistics", "oncology", "Basic cancer research", "knockout", "knockdown", "h460"], "article_id"=>721428, "categories"=>["Biological Sciences", "Medicine", "Mathematics"], "users"=>["Susan Richter", "Shona Morrison", "Tim Connor", "Jiechuang Su", "Cristin G. Print", "Ron S. Ronimus", "Sean L. McGee", "William R. Wilson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0065267.g009", "stats"=>{"downloads"=>0, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Effect_of_knockout_of_i_ADPGK_i_and_its_knockdown_by_siRNA_on_expression_of_i_PPARGC1A_i_in_H460_cells_/721428", "title"=>"Effect of knockout of <i>ADPGK,</i> and its knockdown by siRNA, on expression of <i>PPARGC1A</i> in H460 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-06-14 00:23:48"}
  • {"files"=>["https://ndownloader.figshare.com/files/1088629"], "description"=>"<p><i>HK2</i> was knocked down by siRNA in two independent experiments, combined here, each of which included three biological replicates for WT and two for each knockout clone. (A) HK2 mRNA by qPCR one day after siRNA transfection. (B). Cell number two days after siRNA transfection, at which time cells were replated for clonogenic assay. (C) Plating efficiencies of two days after transfection with control siRNA. (D) Effect of <i>HK2</i> siRNA on clonogenic surviving fraction two days after transfection, relative to cells transfected with control siRNA. (E) Effect of 6 h anoxia on clonogenic surviving fraction, relative to oxic controls, determined by plating cells in an anoxic chamber two days after siRNA transfection and transferring to an aerobic incubator 6 h later. (F) Effect of <i>HK2</i> siRNA on clonogenic surviving fraction after exposure to 6 h anoxia, relative to an equivalent anoxic exposure after control siRNA.</p>", "links"=>[], "tags"=>["Biochemistry", "Bioenergetics", "Energy-producing organelles", "enzymes", "Enzyme metabolism", "metabolism", "Oxygen metabolism", "Computational biology", "microarrays", "genetics", "gene expression", "Gene function", "Genetic mutation", "immunology", "Immunologic techniques", "Immunohistochemical analysis", "Model organisms", "Animal models", "mouse", "Molecular cell biology", "RNA interference", "Cell growth", "statistics", "Biostatistics", "oncology", "Basic cancer research", "h460", "lowers", "clonogenic", "anoxia", "knocked"], "article_id"=>721423, "categories"=>["Biological Sciences", "Medicine", "Mathematics"], "users"=>["Susan Richter", "Shona Morrison", "Tim Connor", "Jiechuang Su", "Cristin G. Print", "Ron S. Ronimus", "Sean L. McGee", "William R. Wilson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0065267.g005", "stats"=>{"downloads"=>0, "page_views"=>20, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Knockout_of_i_ADPGK_i_in_H460_lowers_clonogenic_survival_under_anoxia_and_when_i_HK2_i_is_knocked_down_/721423", "title"=>"Knockout of <i>ADPGK</i> in H460 lowers clonogenic survival under anoxia and when <i>HK2</i> is knocked down.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-06-14 00:23:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/1088630"], "description"=>"<p>Glucose consumption (A, D) and lactate formation (B, E) were measured 2 days after transfection with control siRNA or <i>HK2</i> siRNA. Cells were re-seeded in fresh medium at 2×10<sup>5</sup> per ml in 96-well plates in an anaerobic chamber, and the culture medium was assayed 6 h later. Values are mean and SEM for 3 independent experiments. Asterisks indicate a significant difference for HK2 siRNA relative to control siRNA (p&lt;0.05).</p>", "links"=>[], "tags"=>["Biochemistry", "Bioenergetics", "Energy-producing organelles", "enzymes", "Enzyme metabolism", "metabolism", "Oxygen metabolism", "Computational biology", "microarrays", "genetics", "gene expression", "Gene function", "Genetic mutation", "immunology", "Immunologic techniques", "Immunohistochemical analysis", "Model organisms", "Animal models", "mouse", "Molecular cell biology", "RNA interference", "Cell growth", "statistics", "Biostatistics", "oncology", "Basic cancer research", "suppresses", "anaerobic", "glycolysis", "h460", "knockout"], "article_id"=>721424, "categories"=>["Biological Sciences", "Medicine", "Mathematics"], "users"=>["Susan Richter", "Shona Morrison", "Tim Connor", "Jiechuang Su", "Cristin G. Print", "Ron S. Ronimus", "Sean L. McGee", "William R. Wilson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0065267.g006", "stats"=>{"downloads"=>0, "page_views"=>38, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Knockdown_of_i_HK2_i_suppresses_anaerobic_glycolysis_in_H460_and_HCT116_but_knockout_of_i_ADPGK_i_has_no_effect_/721424", "title"=>"Knockdown of <i>HK2</i> suppresses anaerobic glycolysis in H460 and HCT116, but knockout of <i>ADPGK</i> has no effect.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-06-14 00:23:44"}
  • {"files"=>["https://ndownloader.figshare.com/files/1088628"], "description"=>"<p>RNA was analyzed using Affymetrix Human Gene 1.0 ST microarrays (A) Scatterplot showing mean expression values for HCT116 C3 versus WT (3 cultures each). (B) Scatterplot showing mean expression values for H460 IIE5 versus WT (2 cultures each). (C) Heat map of the 50 top-ranked differentially expressed probe sets in duplicate cultures of H460 WT and H460 IIE3. (D) qPCR of RNA from 3 replicate cell cultures each for H460 WT and <i>ADPGK</i> knockout clones IIE5 and IID10, normalised against 18S rRNA.</p>", "links"=>[], "tags"=>["Biochemistry", "Bioenergetics", "Energy-producing organelles", "enzymes", "Enzyme metabolism", "metabolism", "Oxygen metabolism", "Computational biology", "microarrays", "genetics", "gene expression", "Gene function", "Genetic mutation", "immunology", "Immunologic techniques", "Immunohistochemical analysis", "Model organisms", "Animal models", "mouse", "Molecular cell biology", "RNA interference", "Cell growth", "statistics", "Biostatistics", "oncology", "Basic cancer research", "adpgk-null", "hct116", "h460"], "article_id"=>721422, "categories"=>["Biological Sciences", "Medicine", "Mathematics"], "users"=>["Susan Richter", "Shona Morrison", "Tim Connor", "Jiechuang Su", "Cristin G. Print", "Ron S. Ronimus", "Sean L. McGee", "William R. Wilson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0065267.g004", "stats"=>{"downloads"=>0, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Global_gene_expression_in_ADPGK_null_HCT116_clone_C3_and_H460_clone_IIE5_cell_lines_/721422", "title"=>"Global gene expression in ADPGK-null HCT116 (clone C3) and H460 (clone IIE5) cell lines.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-06-14 00:23:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1088637"], "description"=>"<p>Asterisks indicate significant (p&lt;0.05) difference from WT by one-way ANOVA/Holm-Sidak. Proliferation parameters of respective cell lines are displayed as mean ± SEM for three biological replicates.</p>", "links"=>[], "tags"=>["Biochemistry", "Bioenergetics", "Energy-producing organelles", "enzymes", "Enzyme metabolism", "metabolism", "Oxygen metabolism", "Computational biology", "microarrays", "genetics", "gene expression", "Gene function", "Genetic mutation", "immunology", "Immunologic techniques", "Immunohistochemical analysis", "Model organisms", "Animal models", "mouse", "Molecular cell biology", "RNA interference", "Cell growth", "statistics", "Biostatistics", "oncology", "Basic cancer research", "clonogenicity", "hct116", "h460", "lines"], "article_id"=>721431, "categories"=>["Biological Sciences", "Medicine", "Mathematics"], "users"=>["Susan Richter", "Shona Morrison", "Tim Connor", "Jiechuang Su", "Cristin G. Print", "Ron S. Ronimus", "Sean L. McGee", "William R. Wilson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0065267.t001", "stats"=>{"downloads"=>0, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Proliferation_and_clonogenicity_of_HCT116_and_H460_cell_lines_under_normoxia_/721431", "title"=>"Proliferation and clonogenicity of HCT116 and H460 cell lines under normoxia.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-06-14 00:23:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/1088633"], "description"=>"<p>Basal OCR and ECAR were measured in H460 (A–C) and HCT116 (D–E) <i>ADPGK</i> knockout cell lines and their respective wild types (WT) using a Seahorse XF analyser with monolayers in different media as indicated. Values are mean and SEM from two independent experiments with five biological replicates each.</p>", "links"=>[], "tags"=>["Biochemistry", "Bioenergetics", "Energy-producing organelles", "enzymes", "Enzyme metabolism", "metabolism", "Oxygen metabolism", "Computational biology", "microarrays", "genetics", "gene expression", "Gene function", "Genetic mutation", "immunology", "Immunologic techniques", "Immunohistochemical analysis", "Model organisms", "Animal models", "mouse", "Molecular cell biology", "RNA interference", "Cell growth", "statistics", "Biostatistics", "oncology", "Basic cancer research", "extracellular", "acidification", "knockout", "cells", "aerobic"], "article_id"=>721427, "categories"=>["Biological Sciences", "Medicine", "Mathematics"], "users"=>["Susan Richter", "Shona Morrison", "Tim Connor", "Jiechuang Su", "Cristin G. Print", "Ron S. Ronimus", "Sean L. McGee", "William R. Wilson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0065267.g008", "stats"=>{"downloads"=>0, "page_views"=>34, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Reduced_oxygen_consumption_rate_OCR_but_not_extracellular_acidification_rate_ECAR_in_i_ADPGK_i_knockout_cells_under_aerobic_conditions_/721427", "title"=>"Reduced oxygen consumption rate (OCR) but not extracellular acidification rate (ECAR) in <i>ADPGK</i> knockout cells under aerobic conditions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-06-14 00:23:47"}
  • {"files"=>["https://ndownloader.figshare.com/files/1088617"], "description"=>"<p>(A) Western blots of H460 cells after transfection with ADPGK ZFNs (clones ID5 and VD6), and <i>ADPGK</i>-null clones derived from these in a second round of ZFN transfection (IID10 and IIE5). (B) Genomic DNA copy number by qPCR for primer pairs CNV1-3. Results are plotted relative to WT DNA which has three <i>ADPGK</i> alleles. (C) Sequencing across the ZFN cut site shows deletions (−) and insertions (underlined) for the <i>ADPGK</i> alleles in H460 clones IID10 and IIE5.</p>", "links"=>[], "tags"=>["Biochemistry", "Bioenergetics", "Energy-producing organelles", "enzymes", "Enzyme metabolism", "metabolism", "Oxygen metabolism", "Computational biology", "microarrays", "genetics", "gene expression", "Gene function", "Genetic mutation", "immunology", "Immunologic techniques", "Immunohistochemical analysis", "Model organisms", "Animal models", "mouse", "Molecular cell biology", "RNA interference", "Cell growth", "statistics", "Biostatistics", "oncology", "Basic cancer research", "knockout", "h460", "cells", "zinc-finger"], "article_id"=>721412, "categories"=>["Biological Sciences", "Medicine", "Mathematics"], "users"=>["Susan Richter", "Shona Morrison", "Tim Connor", "Jiechuang Su", "Cristin G. Print", "Ron S. Ronimus", "Sean L. McGee", "William R. Wilson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0065267.g002", "stats"=>{"downloads"=>0, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_i_APDGK_i_knockout_in_H460_cells_using_zinc_finger_nucleases_/721412", "title"=>"<i>APDGK</i> knockout in H460 cells using zinc-finger nucleases.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-06-14 00:23:32"}
  • {"files"=>["https://ndownloader.figshare.com/files/1088614"], "description"=>"<p>(A) Features of the <i>ADPGK</i> gene, with location of primer pairs and the dimeric ZFN targeting site. (B) PCR/CEL-II nuclease (Surveyor) assay for mutation detection at the ZFN target site. HCT116 cells were transfected with a pair of ADPGK ZFNs and a GFP plasmid. One day later genomic DNA was prepared from pooled FACS sorted GFP positive cells for the assay (Lane 4). Lane 1: DNA ladder. Lane 2: positive control DNA from ADPGK ZFN-treated K562 cells. Lane 3: Untreated HCT116 cells. (C) ADPGK ZFN-treated HCT116 cells cloned and screened for <i>ADPGK</i> by western blot. (D) Genomic DNA copy number by qPCR for primer pairs CNV1-3. Results are plotted relative to WT DNA which has two <i>ADPGK</i> alleles. (E) Sequencing across ZFN recognition site (predicted cut site in lower case) resulted in deletions (−) and insertions (underlined) for <i>ADPGK</i>-null HCT116 clones C3 and IC10. The inferred copy number for each allele is shown.</p>", "links"=>[], "tags"=>["Biochemistry", "Bioenergetics", "Energy-producing organelles", "enzymes", "Enzyme metabolism", "metabolism", "Oxygen metabolism", "Computational biology", "microarrays", "genetics", "gene expression", "Gene function", "Genetic mutation", "immunology", "Immunologic techniques", "Immunohistochemical analysis", "Model organisms", "Animal models", "mouse", "Molecular cell biology", "RNA interference", "Cell growth", "statistics", "Biostatistics", "oncology", "Basic cancer research", "knockout", "hct116", "cells", "zinc-finger"], "article_id"=>721409, "categories"=>["Biological Sciences", "Medicine", "Mathematics"], "users"=>["Susan Richter", "Shona Morrison", "Tim Connor", "Jiechuang Su", "Cristin G. Print", "Ron S. Ronimus", "Sean L. McGee", "William R. Wilson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0065267.g001", "stats"=>{"downloads"=>0, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_i_APDGK_i_knockout_in_HCT116_cells_using_zinc_finger_nucleases_/721409", "title"=>"<i>APDGK</i> knockout in HCT116 cells using zinc-finger nucleases.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-06-14 00:23:29"}
  • {"files"=>["https://ndownloader.figshare.com/files/1088632"], "description"=>"<p>ATP was measured 2 days after transfection with control siRNA or <i>HK2</i> siRNA, following re-seeding in fresh medium for 6 h under normoxia (ox) or anoxia (anox). Values are mean and SEM for 3 biological replicates. Asterisks indicate significance (p&lt;0.05) of <i>HK2</i> siRNA treated vs. siRNA control treated cells. Hash indicates p&lt;0.05 for difference between anoxic vs. normoxic conditions.</p>", "links"=>[], "tags"=>["Biochemistry", "Bioenergetics", "Energy-producing organelles", "enzymes", "Enzyme metabolism", "metabolism", "Oxygen metabolism", "Computational biology", "microarrays", "genetics", "gene expression", "Gene function", "Genetic mutation", "immunology", "Immunologic techniques", "Immunohistochemical analysis", "Model organisms", "Animal models", "mouse", "Molecular cell biology", "RNA interference", "Cell growth", "statistics", "Biostatistics", "oncology", "Basic cancer research", "atp", "suppression", "h460", "hct116", "knockout"], "article_id"=>721426, "categories"=>["Biological Sciences", "Medicine", "Mathematics"], "users"=>["Susan Richter", "Shona Morrison", "Tim Connor", "Jiechuang Su", "Cristin G. Print", "Ron S. Ronimus", "Sean L. McGee", "William R. Wilson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0065267.g007", "stats"=>{"downloads"=>1, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Steady_state_ATP_levels_are_more_sensitive_to_i_HK2_i_suppression_in_H460_than_HCT116_cells_but_knockout_of_i_ADPGK_i_has_no_effect_in_either_/721426", "title"=>"Steady state ATP levels are more sensitive to <i>HK2</i> suppression in H460 than HCT116 cells, but knockout of <i>ADPGK</i> has no effect in either.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-06-14 00:23:46"}

PMC Usage Stats | Further Information

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Relative Metric

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