Epidermal Growth-Factor – Induced Transcript Isoform Variation Drives Mammary Cell Migration
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{"title"=>"Epidermal growth-factor - Induced transcript isoform variation drives mammary cell migration", "type"=>"journal", "authors"=>[{"first_name"=>"Wolfgang J.", "last_name"=>"Köstler", "scopus_author_id"=>"57193261244"}, {"first_name"=>"Amit", "last_name"=>"Zeisel", "scopus_author_id"=>"36179097000"}, {"first_name"=>"Cindy", "last_name"=>"Körner", "scopus_author_id"=>"55904975200"}, {"first_name"=>"Jonathan M.", "last_name"=>"Tsai", "scopus_author_id"=>"52564537000"}, {"first_name"=>"Jasmine", "last_name"=>"Jacob-Hirsch", "scopus_author_id"=>"6507065877"}, {"first_name"=>"Nir", "last_name"=>"Ben-Chetrit", "scopus_author_id"=>"18133394900"}, {"first_name"=>"Kirti", "last_name"=>"Sharma", "scopus_author_id"=>"8787631600"}, {"first_name"=>"Hadas", "last_name"=>"Cohen-Dvashi", "scopus_author_id"=>"55432956600"}, {"first_name"=>"Assif", "last_name"=>"Yitzhaky", "scopus_author_id"=>"8834483500"}, {"first_name"=>"Eric", "last_name"=>"Lader", "scopus_author_id"=>"6701692004"}, {"first_name"=>"Ulrich", "last_name"=>"Tschulena", "scopus_author_id"=>"9737837700"}, {"first_name"=>"Gideon", "last_name"=>"Rechavi", "scopus_author_id"=>"7004955283"}, {"first_name"=>"Eytan", "last_name"=>"Domany", "scopus_author_id"=>"7006024239"}, {"first_name"=>"Stefan", "last_name"=>"Wiemann", "scopus_author_id"=>"7004272878"}, {"first_name"=>"Yosef", "last_name"=>"Yarden", "scopus_author_id"=>"7004959601"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "scopus"=>"2-s2.0-84891942332", "pmid"=>"24324612", "doi"=>"10.1371/journal.pone.0080566", "pui"=>"372094592", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "sgr"=>"84891942332"}, "id"=>"e2be09b1-27ee-3223-8e85-5fe422c17945", "abstract"=>"Signal-induced transcript isoform variation (TIV) includes alternative promoter usage as well as alternative splicing and alternative polyadenylation of mRNA. To assess the phenotypic relevance of signal-induced TIV, we employed exon arrays and breast epithelial cells, which migrate in response to the epidermal growth factor (EGF). We show that EGF rapidly--within one hour--induces widespread TIV in a significant fraction of the transcriptome. Importantly, TIV characterizes many genes that display no differential expression upon stimulus. In addition, similar EGF-dependent changes are shared by a panel of mammary cell lines. A functional screen, which utilized isoform-specific siRNA oligonucleotides, indicated that several isoforms play essential, non-redundant roles in EGF-induced mammary cell migration. Taken together, our findings highlight the importance of TIV in the rapid evolvement of a phenotypic response to extracellular signals.", "link"=>"http://www.mendeley.com/research/epidermal-growthfactor-induced-transcript-isoform-variation-drives-mammary-cell-migration", "reader_count"=>24, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>3, "Researcher"=>4, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>10, "Other"=>1, "Student > Bachelor"=>4, "Professor"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>3, "Researcher"=>4, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>10, "Other"=>1, "Student > Bachelor"=>4, "Professor"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>6, "Agricultural and Biological Sciences"=>13, "Medicine and Dentistry"=>2, "Neuroscience"=>1, "Computer Science"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Neuroscience"=>{"Neuroscience"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>13}, "Computer Science"=>{"Computer Science"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>6}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"China"=>1}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1306719"], "description"=>"<p>(<b>A</b>) <i><u>Experimental design:</u></i> Biological triplicates of MCF10A cells were stimulated with EGF (20 ng/ml) for the indicated intervals and each sample was hybridized separately to Affymetrix Human Exon 1.0 arrays. (<b>B</b>) Analysis outlines. <i><u>Upper panel:</u></i> For each gene, both the annotation and probe set (PS) information were used to define the PS as interrogating either constitutive introns (red) or putative exons (blue). <i><u>Middle panel:</u></i> Only putative exonic PS whose signal intensities were significantly higher than the introns' were considered. <i><u>Lower panel:</u></i> At each time point, differentially expressed genes were identified as follows: Fold change (FC) of the signal intensity between unstimulated and stimulated cells was calculated for all exonic PS. Gene-level FC (dashed line) is the median exonic FC. EGF-induced TIV events were identified whenever FC of exonic PS behaved differently from the gene-level FC; statistical significance was assessed using FDR analysis. (<b>C</b>) Venn diagrams showing the number of significantly (FDR<5%) differentially expressed genes, with FC ≥1.5, along with the number of genes for which TIV took place (FDR<5%) at two or more adjacent time points. One gene, <i>WEE1</i>, was found to be significantly up-regulated at early time points following EGF stimulation and down-regulated towards the end of the time course. (<b>D</b>) Histograms showing the number of TIV events present at different time points and the cumulative number of events following an EGF stimulus, separately for different types of TIV events. For all analyses, only TIV events detectable in at least two adjacent time points were considered. Hence, TIV events detectable 480 min after stimulation are not displayed because they had to be present at 240 minutes as well.</p>", "links"=>[], "tags"=>["time-dependent", "tiv", "mammary"], "article_id"=>870957, "categories"=>["Biological Sciences"], "users"=>["Wolfgang J. Köstler", "Amit Zeisel", "Cindy Körner", "Jonathan M. Tsai", "Jasmine Jacob-Hirsch", "Nir Ben-Chetrit", "Kirti Sharma", "Hadas Cohen-Dvashi", "Assif Yitzhaky", "Eric Lader", "Ulrich Tschulena", "Gideon Rechavi", "Eytan Domany", "Stefan Wiemann", "Yosef Yarden"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0080566.g001", "stats"=>{"downloads"=>0, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_EGF_induces_time_dependent_TIV_in_mammary_cells_/870957", "title"=>"EGF induces time-dependent TIV in mammary cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-06 03:14:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/1306747", "https://ndownloader.figshare.com/files/1306748", "https://ndownloader.figshare.com/files/1306749", "https://ndownloader.figshare.com/files/1306750", "https://ndownloader.figshare.com/files/1306751", "https://ndownloader.figshare.com/files/1306752", "https://ndownloader.figshare.com/files/1306753", "https://ndownloader.figshare.com/files/1306754", "https://ndownloader.figshare.com/files/1306755"], "description"=>"<div><p>Signal-induced transcript isoform variation (TIV) includes alternative promoter usage as well as alternative splicing and alternative polyadenylation of mRNA. To assess the phenotypic relevance of signal-induced TIV, we employed exon arrays and breast epithelial cells, which migrate in response to the epidermal growth factor (EGF). We show that EGF rapidly – within one hour – induces widespread TIV in a significant fraction of the transcriptome. Importantly, TIV characterizes many genes that display no differential expression upon stimulus. In addition, similar EGF-dependent changes are shared by a panel of mammary cell lines. A functional screen, which utilized isoform-specific siRNA oligonucleotides, indicated that several isoforms play essential, non-redundant roles in EGF-induced mammary cell migration. Taken together, our findings highlight the importance of TIV in the rapid evolvement of a phenotypic response to extracellular signals.</p></div>", "links"=>[], "tags"=>["growth-factor", "induced", "transcript", "isoform", "drives", "mammary"], "article_id"=>870984, "categories"=>["Biological Sciences"], "users"=>["Wolfgang J. Köstler", "Amit Zeisel", "Cindy Körner", "Jonathan M. Tsai", "Jasmine Jacob-Hirsch", "Nir Ben-Chetrit", "Kirti Sharma", "Hadas Cohen-Dvashi", "Assif Yitzhaky", "Eric Lader", "Ulrich Tschulena", "Gideon Rechavi", "Eytan Domany", "Stefan Wiemann", "Yosef Yarden"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0080566.s001", "https://dx.doi.org/10.1371/journal.pone.0080566.s002", "https://dx.doi.org/10.1371/journal.pone.0080566.s003", "https://dx.doi.org/10.1371/journal.pone.0080566.s004", "https://dx.doi.org/10.1371/journal.pone.0080566.s005", "https://dx.doi.org/10.1371/journal.pone.0080566.s006", "https://dx.doi.org/10.1371/journal.pone.0080566.s007", "https://dx.doi.org/10.1371/journal.pone.0080566.s008", "https://dx.doi.org/10.1371/journal.pone.0080566.s009"], "stats"=>{"downloads"=>4, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Epidermal_Growth_Factor_8211_Induced_Transcript_Isoform_Variation_Drives_Mammary_Cell_Migration_/870984", "title"=>"Epidermal Growth-Factor – Induced Transcript Isoform Variation Drives Mammary Cell Migration", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-12-06 03:14:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/1306727"], "description"=>"<p>(<b>A</b>) <i><u>Upper panel:</u></i> Organization of <i>LAMA3</i> transcripts as defined by the UCSC gene model. Vertical boxes represent exons; the arrow (100 kb) indicates the direction of transcription. Positions of exonic PS (blue) that passed all filtering steps are indicated. The upmost isoform was not expressed in MCF10A cells (neither of two isoform-specific PS interrogating the alternative last exon passed the required signal intensity threshold). <i><u>Lower panels:</u></i> FC (log2 scale) of each PS with respect to pre-stimulus values is shown for each time point. Only PS with ‘present’ calls in all replicates are depicted at each time point. Bars represent standard errors of FC from three biological replicates. Note strong induction of the 3′ portion (the dashed green line marks the median FC of this region). In contrast, the longer isoforms (median FC shown by the dashed red line) are slightly downregulated. (<b>B</b>) The heat map displays the FC ratios of the short to long isoforms of the top 40 genes identified as undergoing EGF-induced alternative usage of well-annotated promoters (FDR<1%) resulting in alternative first exons; if the relative abundance of the short isoforms compared to the long isoforms increases after EGF stimulation the FC ratio becomes ‘high’ (see for instance <i>LAMA3</i>) (<b>C</b>) For qPCR validation of TIV events primer pairs specific to transcript isoforms were used for each gene, along with pairs spanning regions common to all transcripts. Error bars represent standard deviations from three technical replicates. The experiment was repeated twice.</p>", "links"=>[], "tags"=>["promoter"], "article_id"=>870965, "categories"=>["Biological Sciences"], "users"=>["Wolfgang J. Köstler", "Amit Zeisel", "Cindy Körner", "Jonathan M. Tsai", "Jasmine Jacob-Hirsch", "Nir Ben-Chetrit", "Kirti Sharma", "Hadas Cohen-Dvashi", "Assif Yitzhaky", "Eric Lader", "Ulrich Tschulena", "Gideon Rechavi", "Eytan Domany", "Stefan Wiemann", "Yosef Yarden"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0080566.g002", "stats"=>{"downloads"=>1, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_EGF_induced_alternative_promoter_usage_/870965", "title"=>"EGF-induced alternative promoter usage.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-06 03:14:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/1306729"], "description"=>"<p>(<b>A</b>) For 35 genes, each transcript isoform undergoing EGF-induced TIV was targeted by two individual oligonucleotides and by ‘gene-level’ siRNA oligonucleotide pools knocking down all isoforms. An automated robotic platform was used for performing scratches. Each candidate was screened in eleven biological replicates. (<b>B</b>) Scheme of the automated image analysis used to calculate the average migration distance (AMD) for each well compared to control siRNA oligonucleotides (<b>C</b>) <i><u>Upper panel:</u></i> Each bar represents the measured mean relative AMD (and standard error) for one oligonucleotide. <i><u>Lower panel:</u></i> The FDR q-values, relative to the control siRNA, are shown.</p>", "links"=>[], "tags"=>["egf-induced", "tiv", "isoform-specific", "sirna"], "article_id"=>870967, "categories"=>["Biological Sciences"], "users"=>["Wolfgang J. Köstler", "Amit Zeisel", "Cindy Körner", "Jonathan M. Tsai", "Jasmine Jacob-Hirsch", "Nir Ben-Chetrit", "Kirti Sharma", "Hadas Cohen-Dvashi", "Assif Yitzhaky", "Eric Lader", "Ulrich Tschulena", "Gideon Rechavi", "Eytan Domany", "Stefan Wiemann", "Yosef Yarden"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0080566.g003", "stats"=>{"downloads"=>0, "page_views"=>25, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Functional_tests_of_EGF_induced_TIV_by_an_isoform_specific_siRNA_screen_/870967", "title"=>"Functional tests of EGF-induced TIV by an isoform-specific siRNA screen.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-06 03:14:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/1306730"], "description"=>"<p>(<b>A</b>) MCF10A cells were transfected with siRNA oligonucleotides (20 nM) targeting individual isoforms of <i>LAMA3</i> (left panel) or <i>PTHLH</i> (right panel), siRNA pools targeting all isoforms (‘gene-level’) or with control scrambled siRNA oligonucleotides (sictrl denotes siRNA control, ‘i1,o1’ denotes the first oligonucleotide targeting isoform 1; ‘i2,o2’ the second oligonucleotide targeting isoform 2, etc. and ‘GL’ denotes ‘gene level’). Forty-eight hours later, RNA was extracted and qPCR measurements were performed using primers specific to all expressed individual isoforms, as well as primers amplifying transcript regions common to all expressed isoforms. Measurements were normalized to results obtained with an independent siRNA control replicate. Error bars denote standard errors from three technical replicates and asterisks denote statistically significant (p<0.05) differences relative to siCtrl. Similar results were obtained in three independent repeat experiments. (<b>B</b>) Oligonucleotides for <i>EGFR</i>, which impair migration, and for <i>CSNK1G2</i>, which accelerate migration are shown as additional controls. Cell cycle analysis was performed as described in <i>Methods</i>. Error bars represent the standard errors from the analysis of 24 images taken per from each of four wells per condition. (<b>C</b>) Viability was measured by tetrazolium-based WST-1 assay. MCF10A cells were transfected with the indicated oligonucleotides (siRNA to polo-like kinase, <i>PLK1</i>, served as positive control). Error bars represent standard errors from 3 biological replicates. The experiment was performed thrice. (<b>D</b>) Shown are morphologic effects of isoform-specific oligonucleotides targeting <i>LAMA3</i> or <i>PTHLH</i>. Nine thousand MCF10A cells per well were transfected, starved and stimulated as described for the proliferation assays, followed by DAPI and phalloidin staining, 14 hours after stimulation. Automated image analysis was used to systematically assess changes in cell size and shape by analyzing 24 images taken per condition. No difference between the individual knockdowns was apparent (see also <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080566#pone.0080566.s006\" target=\"_blank\">Figure S6B</a>); one representative image for each condition is shown.</p>", "links"=>[], "tags"=>["knockdown", "viability", "morphological"], "article_id"=>870968, "categories"=>["Biological Sciences"], "users"=>["Wolfgang J. Köstler", "Amit Zeisel", "Cindy Körner", "Jonathan M. Tsai", "Jasmine Jacob-Hirsch", "Nir Ben-Chetrit", "Kirti Sharma", "Hadas Cohen-Dvashi", "Assif Yitzhaky", "Eric Lader", "Ulrich Tschulena", "Gideon Rechavi", "Eytan Domany", "Stefan Wiemann", "Yosef Yarden"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0080566.g004", "stats"=>{"downloads"=>0, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Isoform_specific_knockdown_and_absence_of_significant_proliferation_viability_and_morphological_effects_/870968", "title"=>"Isoform-specific knockdown and absence of significant proliferation, viability and morphological effects.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-06 03:14:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/1306738"], "description"=>"<p>(<b>A</b>) Time lapse microscopy images (one of 5 repeats) of scratch assays using MCF10A cells transfected with isoform-specific siRNAs to <i>PTHLH</i> (left) and quantification of all images (right). Error bars represent the standard deviations of five repeats per condition; asterisks indicate significant differences from siCONTROL (*p<0.05, **p<0.01). (<b>B</b> and <b>C</b>) Results of Real-time Cell Analyzer (RTCA) experiments measuring the effects of isoform-specific oligonucleotides to <i>PTHLH</i> (B) or to <i>LAMA3</i> (C) on cell adhesion (left panels) and single cell migration through filters (right panels). Oligonucleotides targeting transcript regions common to all expressed isoforms of the respective gene were also employed (si gene-level). Cells treated with transfection reagent alone (‘mock’) and cells transfected with scrambled control siRNA oligonucleotides (siCONTROL) are shown as controls. Error bars represent the standard deviations of 3 or more repeats per condition; asterisks indicate significant differences relative to siCONTROL (*p<0.05, **p<0.01). The experiment was repeated thrice.</p>", "links"=>[], "tags"=>["hits", "sirna"], "article_id"=>870975, "categories"=>["Biological Sciences"], "users"=>["Wolfgang J. Köstler", "Amit Zeisel", "Cindy Körner", "Jonathan M. Tsai", "Jasmine Jacob-Hirsch", "Nir Ben-Chetrit", "Kirti Sharma", "Hadas Cohen-Dvashi", "Assif Yitzhaky", "Eric Lader", "Ulrich Tschulena", "Gideon Rechavi", "Eytan Domany", "Stefan Wiemann", "Yosef Yarden"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0080566.g005", "stats"=>{"downloads"=>0, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Validation_of_hits_of_the_siRNA_screen_/870975", "title"=>"Validation of hits of the siRNA screen.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-06 03:14:49"}

PMC Usage Stats | Further Information

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Relative Metric

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