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{"title"=>"SOX4 Mediates TGF-β-Induced Expression of Mesenchymal Markers during Mammary Cell Epithelial to Mesenchymal Transition", "type"=>"journal", "authors"=>[{"first_name"=>"Stephin J.", "last_name"=>"Vervoort", "scopus_author_id"=>"55230021500"}, {"first_name"=>"Ana Rita", "last_name"=>"Lourenço", "scopus_author_id"=>"55545619800"}, {"first_name"=>"Ruben", "last_name"=>"van Boxtel", "scopus_author_id"=>"25633147400"}, {"first_name"=>"Paul J.", "last_name"=>"Coffer", "scopus_author_id"=>"7006018224"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"pmid"=>"23301048", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "doi"=>"10.1371/journal.pone.0053238", "issn"=>"19326203", "scopus"=>"2-s2.0-84871858845", "pui"=>"368044665", "sgr"=>"84871858845"}, "id"=>"a4ae9e35-d3a0-3677-8491-4661076f4374", "abstract"=>"The epithelial to mensenchymal transition program regulates various aspects of embryonic development and tissue homeostasis, but aberrant activation of this pathway in cancer contributes to tumor progression and metastasis. TGF-b potently induces an epithelial to mensenchymal transition in cancers of epithelial origin by inducing transcriptional changes mediated by several key transcription factors. Here, we identify the developmental transcription factor SOX4 as a transcriptional target of TGF-b in immortalized human mammary epithelial cells. SOX4 expression and activity are rapidly induced in the early stages of the TGF-b-induced epithelial to mensenchymal transition. We demonstrate that conditional activation of Sox4 is sufficient to induce the expression of N-cadherin and additional mesenchymal markers including vimentin and fibronectin, but fails to induce complete EMT as no changes are observed in the expression of E-cadherin and b-catenin. Moreover, shRNA-mediated knockdown of SOX4 significantly delays TGF-b-induced mRNA and protein expression of mesenchymal markers. Taken together, these data suggest that TGF-b-mediated increased expression of SOX4 is required for the induction of a mesenchymal phenotype during EMT in human mammary epithelial cells.", "link"=>"http://www.mendeley.com/research/sox4-mediates-tgf%CE%B2induced-expression-mesenchymal-markers-during-mammary-cell-epithelial-mesenchymal", "reader_count"=>44, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>2, "Researcher"=>11, "Student > Doctoral Student"=>4, "Student > Ph. D. Student"=>11, "Student > Postgraduate"=>1, "Student > Master"=>10, "Student > Bachelor"=>1, "Lecturer"=>1, "Professor"=>1, "Other"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>2, "Researcher"=>11, "Student > Doctoral Student"=>4, "Student > Ph. D. Student"=>11, "Student > Postgraduate"=>1, "Student > Master"=>10, "Student > Bachelor"=>1, "Lecturer"=>1, "Professor"=>1, "Other"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>2, "Environmental Science"=>1, "Biochemistry, Genetics and Molecular Biology"=>7, "Agricultural and Biological Sciences"=>25, "Medicine and Dentistry"=>8, "Chemistry"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>8}, "Chemistry"=>{"Chemistry"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>25}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>7}, "Unspecified"=>{"Unspecified"=>2}, "Environmental Science"=>{"Environmental Science"=>1}}, "reader_count_by_country"=>{"Republic of Singapore"=>1, "Austria"=>1, "Sweden"=>1, "United States"=>1, "Germany"=>1}, "group_count"=>0}

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Figshare

• {"files"=>["https://ndownloader.figshare.com/files/513668"], "description"=>"<p>(<b>A</b>) MARA analysis predicts Sox activity during EMT in HMEC and NMuMG cells (see text for details) (<b>B</b>) Public microarrays databases generated in non-transformed HMEC cells treated with TGF-β or left untreated were analyzed and SOX4 expression was assessed. (<b>C</b>) HMLE cells were stimulated with 2.5 ng/mL of TGF-β as indicated, lysed and mRNA expression of <i>CDH2</i> (N-cadherin), <i>VIM</i> (vimentin), <i>CDH1</i> (E-cadherin) were analyzed by qRT-PCR. (<b>D</b>) HMLE cells were stimulated with 2.5 ng/mL of TGF-β as indicated and lysed. The protein lysates were visualized by Western-Blotting using anti-N-cadherin, anti-SOX4, anti-Tubulin and anti-E-cadherin antibodies. Western blot data is representative of at least three independent experiments. *p<0,05 (N = 3±SD).</p>", "links"=>[], "tags"=>["genetics and genomics", "molecular biology", "Computational biology", "oncology", "biophysics", "developmental biology", "Biochemistry"], "article_id"=>184155, "categories"=>["Molecular Biology", "Biochemistry", "Developmental Biology", "Biophysics", "Cancer", "Biological Sciences", "Genetics"], "users"=>["Stephin J. Vervoort", "Ana Rita Lourenço", "Ruben van Boxtel", "Paul J. Coffer"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0053238.g001", "stats"=>{"downloads"=>1, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_SOX4_expression_is_increased_by_TGF_946_during_EMT_/184155", "title"=>"SOX4 expression is increased by TGF-β during EMT.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-01-03 01:09:15"}
• {"files"=>["https://ndownloader.figshare.com/files/513914"], "description"=>"<p>(<b>A</b>) HMLE cell lines expressing ER:Sox4 were stimulated with 4-OHT (100 mM) as indicated. Cells were lysed and mRNA expression of <i>CDH2</i> (N-cadherin), <i>VIM</i> (vimentin), <i>FN1</i> (fibronectin) and <i>CDH1</i> (E-cadherin) was analyzed by qRT-PCR. (<b>B</b>) HEK293T cells were transiently transfected with Flag-tagged Sox4 Wt or Flag-tagged Sox4 1-135aa and co-transfected with a <i>CDH2</i> luciferase reporter construct as indicated. After 48 hours luciferase activity was measured. Protein expression was assayed by Western blotting using anti-Flag antibody. (<b>C</b>) Schematic representation of the <i>CDH2</i> promoter region and predicted Sox4 binding sites. (<b>D</b>) Chromatin Immunoprecipitation (ChIP) assay using IgG and SOX4 antibodies in MDA-MB-231 cells. Real time PCR was performed using <i>CDH2</i> promoter-specific primers to test SOX4 occupancy at this region. (<b>E</b>) HEK293T cells were transiently transfected with the empty vector pcDNA3 or Flag-tagged Sox4 Wt. After 48 hours cells were harvested and nuclear fraction was extracted. Nuclear extracts were used to perform a biotinylated oligonucleotide pull down assay in which three <i>CDH2</i> promoter sites and two sites localized in the first intron of <i>CDH2</i> were included. Lysates were assessed by western blotting using anti-Flag antibody. (<b>F</b>) HMLE cell lines expressing ER:Sox4 were stimulated with 4-OHT (100 nM) as indicated or left untreated. Cells were lysed and lysates were analyzed by Western blotting using anti-N-cadherin, anti-Tubulin, anti-E-cadherin and anti-ER antibodies. (<b>G</b>) HMLE cells expressing ER:Sox4 were treated with 4-OHT (100 nM) as indicated. Cells were fixed, permeabilized and the expression of N-cadherin and E-cadherin was assessed (green and red respectively). Blue = DAPI. Western blot and confocal microscopy data is representative of at least three independent experiments. *p<0,05 (N = 3±SD).</p>", "links"=>[], "tags"=>["activation", "induces", "upregulation", "mesenchymal"], "article_id"=>184413, "categories"=>["Molecular Biology", "Biochemistry", "Developmental Biology", "Biophysics", "Cancer", "Biological Sciences", "Genetics"], "users"=>["Stephin J. Vervoort", "Ana Rita Lourenço", "Ruben van Boxtel", "Paul J. Coffer"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0053238.g003", "stats"=>{"downloads"=>1, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Sox4_activation_induces_upregulation_of_mesenchymal_markers_/184413", "title"=>"Sox4 activation induces upregulation of mesenchymal markers.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-01-03 01:13:33"}
• {"files"=>["https://ndownloader.figshare.com/files/513770"], "description"=>"<p>The hormone binding domain of the ER was fused to the N-terminus of Sox4. (<b>A</b>) ER:<i>Sox4</i> was stably transduced in U2OS cells. Immuno-fluorescence analysis of ER:Sox4 localization using an anti-ER antibody after stimulation with 4-OHT (100 nM) for the time points indicated. (<b>B</b>) U2OS cells expressing ER and ER:Sox4 were transfected with an optimal Sox4 luciferase reporter construct and treated overnight with 4-OHT (100 nM) after which luciferase activity was measured. (<b>C</b>) ER:Sox4 localization in HMLE cells in presence and absence of 4-OHT. (<b>D</b>) HMLE cells expressing ER and ER:Sox4 were transfected with an optimal Sox4 luciferase reporter construct and stimulated overnight with 4-OHT (100 nM) after which luciferase activity was measured. Confocal microscopy data is representative of at least three independent experiments. *p<0,05 (N = 3±SD).</p>", "links"=>[], "tags"=>["sox4", "conditional", "activation"], "article_id"=>184264, "categories"=>["Molecular Biology", "Biochemistry", "Developmental Biology", "Biophysics", "Cancer", "Biological Sciences", "Genetics"], "users"=>["Stephin J. Vervoort", "Ana Rita Lourenço", "Ruben van Boxtel", "Paul J. Coffer"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0053238.g002", "stats"=>{"downloads"=>0, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Generation_of_a_Sox4_conditional_activation_system_/184264", "title"=>"Generation of a Sox4 conditional activation system.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-01-03 01:11:04"}
• {"files"=>["https://ndownloader.figshare.com/files/514153"], "description"=>"<p>Transcription factors are indicated in bold.</p>", "links"=>[], "tags"=>["ontology", "dna-dependent", "genes", "regulated", "greater", "upregulated"], "article_id"=>184646, "categories"=>["Molecular Biology", "Biochemistry", "Developmental Biology", "Biophysics", "Cancer", "Biological Sciences", "Genetics"], "users"=>["Stephin J. Vervoort", "Ana Rita Lourenço", "Ruben van Boxtel", "Paul J. Coffer"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0053238.t004", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Gene_Ontology_category_positive_regulation_of_transcription_DNA_dependent_GO_0045893_genes_significantly_regulated_and_greater_than_2_fold_upregulated_during_TGF_946_induced_EMT_/184646", "title"=>"Gene Ontology category ''positive regulation of transcription, DNA-dependent (GO:0045893) genes significantly regulated and greater than 2 fold upregulated during TGF-β-induced EMT.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-01-03 01:17:26"}
• {"files"=>["https://ndownloader.figshare.com/files/514035"], "description"=>"<p>HMLE cells line were either transduced with a control shRNA (Scr shRNA) or with a shRNA targeting SOX4 (SOX4 shRNA). (<b>A</b>) Scr shRNA and SOX4 shRNA cell lines were either treated with 2.5 ng/mL of TGF-β for 7 days or left untreated. Cells were lysed and analyzed by Western Blotting using anti-SOX4 and anti-Tubulin antibodies (<b>B</b>) HMLE cell lines expressing Scr shRNA and SOX4 shRNA were stimulated with 2.5 ng/mL of TGF-β as indicated. Cells were lysed and mRNA expression of <i>CDH2</i> (N-cadherin), <i>VIM</i> (vimentin) and <i>CDH1</i> (E-cadherin) were assessed. (<b>C</b>) Cell lysates of HMLE cell lines expressing Scr shRNA and SOX4 shRNA stimulated with 2.5 ng/mL of TGF-β as indicated and analyzed by western blotting using N-cadherin, anti-Tubulin and anti-E-cadherin antibodies. (<b>D</b>) Scr shRNA and SOX4 shRNA cell lines were stimulated with 2.5 ng/mL of TGF-β as indicated. Cells were fixed, permeabilized and N-cadherin expression was assessed (green). Blue = DAPI; red = phallodin. Western blot and confocal microscopy data is representative of at least three independent experiments. *p<0,05 (N = 3±SD).</p>", "links"=>[], "tags"=>["knockdown", "delays"], "article_id"=>184532, "categories"=>["Molecular Biology", "Biochemistry", "Developmental Biology", "Biophysics", "Cancer", "Biological Sciences", "Genetics"], "users"=>["Stephin J. Vervoort", "Ana Rita Lourenço", "Ruben van Boxtel", "Paul J. Coffer"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0053238.g004", "stats"=>{"downloads"=>1, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_SOX4_knockdown_delays_TGF_946_induced_EMT_/184532", "title"=>"SOX4 knockdown delays TGF-β-induced EMT.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-01-03 01:15:32"}
• {"files"=>["https://ndownloader.figshare.com/files/514229"], "description"=>"<p>qRT-PCR primer sequences used in the biotinylated oligonucleotide pull down assay.</p>", "links"=>[], "tags"=>["primer", "sequences", "biotinylated", "oligonucleotide"], "article_id"=>184721, "categories"=>["Molecular Biology", "Biochemistry", "Developmental Biology", "Biophysics", "Cancer", "Biological Sciences", "Genetics"], "users"=>["Stephin J. Vervoort", "Ana Rita Lourenço", "Ruben van Boxtel", "Paul J. Coffer"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0053238.t003", "stats"=>{"downloads"=>4, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_qRT_PCR_primer_sequences_used_in_the_biotinylated_oligonucleotide_pull_down_assay_/184721", "title"=>"qRT-PCR primer sequences used in the biotinylated oligonucleotide pull down assay.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-01-03 01:18:41"}
• {"files"=>["https://ndownloader.figshare.com/files/514253"], "description"=>"<p>Antibodies conditions for western blot analysis.</p>", "links"=>[], "tags"=>["conditions", "blot"], "article_id"=>184752, "categories"=>["Molecular Biology", "Biochemistry", "Developmental Biology", "Biophysics", "Cancer", "Biological Sciences", "Genetics"], "users"=>["Stephin J. Vervoort", "Ana Rita Lourenço", "Ruben van Boxtel", "Paul J. Coffer"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0053238.t002", "stats"=>{"downloads"=>1, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Antibodies_conditions_for_western_blot_analysis_/184752", "title"=>"Antibodies conditions for western blot analysis.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-01-03 01:19:12"}
• {"files"=>["https://ndownloader.figshare.com/files/279758", "https://ndownloader.figshare.com/files/279814"], "description"=>"<div><p>The epithelial to mensenchymal transition program regulates various aspects of embryonic development and tissue homeostasis, but aberrant activation of this pathway in cancer contributes to tumor progression and metastasis. TGF-β potently induces an epithelial to mensenchymal transition in cancers of epithelial origin by inducing transcriptional changes mediated by several key transcription factors. Here, we identify the developmental transcription factor <em>SOX4</em> as a transcriptional target of TGF-β in immortalized human mammary epithelial cells. SOX4 expression and activity are rapidly induced in the early stages of the TGF-β-induced epithelial to mensenchymal transition. We demonstrate that conditional activation of Sox4 is sufficient to induce the expression of N-cadherin and additional mesenchymal markers including vimentin and fibronectin, but fails to induce complete EMT as no changes are observed in the expression of E-cadherin and β-catenin. Moreover, shRNA-mediated knockdown of SOX4 significantly delays TGF-β-induced mRNA and protein expression of mesenchymal markers. Taken together, these data suggest that TGF-β-mediated increased expression of SOX4 is required for the induction of a mesenchymal phenotype during EMT in human mammary epithelial cells.</p> </div>", "links"=>[], "tags"=>["sox4", "mediates", "mesenchymal", "markers", "mammary", "epithelial", "transition"], "article_id"=>115165, "categories"=>["Molecular Biology", "Biochemistry", "Developmental Biology", "Biophysics", "Cancer", "Biological Sciences", "Genetics"], "users"=>["Stephin J. Vervoort", "Ana Rita Lourenço", "Ruben van Boxtel", "Paul J. Coffer"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0053238.s001", "https://dx.doi.org/10.1371/journal.pone.0053238.s002"], "stats"=>{"downloads"=>2, "page_views"=>18, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/SOX4_Mediates_TGF_Induced_Expression_of_Mesenchymal_Markers_during_Mammary_Cell_Epithelial_to_Mesenchymal_Transition__/115165", "title"=>"SOX4 Mediates TGF-β-Induced Expression of Mesenchymal Markers during Mammary Cell Epithelial to Mesenchymal Transition", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-01-03 01:26:05"}
• {"files"=>["https://ndownloader.figshare.com/files/514189"], "description"=>"<p>qRT-PCR primers.</p>", "links"=>[], "tags"=>["genetics and genomics", "molecular biology", "Computational biology", "oncology", "biophysics", "developmental biology", "Biochemistry"], "article_id"=>184678, "categories"=>["Molecular Biology", "Biochemistry", "Developmental Biology", "Biophysics", "Cancer", "Biological Sciences", "Genetics"], "users"=>["Stephin J. Vervoort", "Ana Rita Lourenço", "Ruben van Boxtel", "Paul J. Coffer"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0053238.t001", "stats"=>{"downloads"=>1, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_qRT_PCR_primers_/184678", "title"=>"qRT-PCR primers.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-01-03 01:17:58"}

PMC Usage Stats | Further Information

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Relative Metric

{"start_date"=>"2013-01-01T00:00:00Z", "end_date"=>"2013-12-31T00:00:00Z", "subject_areas"=>[{"subject_area"=>"/Biology and life sciences", "average_usage"=>[269, 466, 588, 697, 800, 896, 988, 1076, 1165, 1254, 1340, 1417]}, {"subject_area"=>"/Biology and life sciences/Biochemistry", "average_usage"=>[266, 468, 593, 703, 804, 903, 993, 1084, 1171, 1256, 1339, 1422, 1492]}, {"subject_area"=>"/Medicine and health sciences", "average_usage"=>[264, 460, 584, 692, 794, 887, 978, 1067, 1154, 1241, 1328, 1408, 1474]}, {"subject_area"=>"/Medicine and health sciences/Oncology", "average_usage"=>[249, 468, 599, 718, 820, 920, 1008, 1093, 1181, 1281, 1357, 1444, 1517]}]}

F1000Prime