Translational Up-Regulation and High-Level Protein Expression from Plasmid Vectors by mTOR Activation via Different Pathways in PC3 and 293T Cells
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{"title"=>"Translational up-regulation and high-level protein expression from plasmid vectors by mTOR activation via different pathways in PC3 and 293T cells", "type"=>"journal", "authors"=>[{"first_name"=>"Prashanthi", "last_name"=>"Karyala", "scopus_author_id"=>"7403016598"}, {"first_name"=>"Nima D.", "last_name"=>"Namsa", "scopus_author_id"=>"29067967100"}, {"first_name"=>"Durga Rao", "last_name"=>"Chilakalapudi", "scopus_author_id"=>"36717306700"}], "year"=>2010, "source"=>"PLoS ONE", "identifiers"=>{"pui"=>"361034511", "doi"=>"10.1371/journal.pone.0014408", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pmid"=>"21203441", "issn"=>"19326203", "scopus"=>"2-s2.0-78650811424", "sgr"=>"78650811424"}, "id"=>"0781abcc-27fd-3110-816e-04652b11268d", "abstract"=>"BACKGROUND: Though 293T cells are widely used for expression of proteins from transfected plasmid vectors, the molecular basis for the high-level expression is yet to be understood. We recently identified the prostate carcinoma cell line PC3 to be as efficient as 293T in protein expression. This study was undertaken to decipher the molecular basis of high-level expression in these two cell lines.\\n\\nMETHODOLOGY/PRINCIPAL FINDINGS: In a survey of different cell lines for efficient expression of platelet-derived growth factor-B (PDGF-B), β-galactosidase (β-gal) and green fluorescent protein (GFP) from plasmid vectors, PC3 was found to express at 5-50-fold higher levels compared to the bone metastatic prostate carcinoma cell line PC3BM and many other cell lines. Further, the efficiency of transfection and level of expression of the reporters in PC3 were comparable to that in 293T. Comparative analyses revealed that the high level expression of the reporters in the two cell lines was due to increased translational efficiency. While phosphatidic acid (PA)-mediated activation of mTOR, as revealed by drastic reduction in reporter expression by n-butanol, primarily contributed to the high level expression in PC3, multiple pathways involving PA, PI3K/Akt and ERK1/2 appear to contribute to the abundant reporter expression in 293T. Thus the extent of translational up-regulation attained through the concerted activation of mTOR by multiple pathways in 293T could be achieved through its activation primarily by the PA pathway in PC3.\\n\\nCONCLUSIONS/SIGNIFICANCE: Our studies reveal that the high-level expression of proteins from plasmid vectors is effected by translational up-regulation through mTOR activation via different signaling pathways in the two cell lines and that PC3 is as efficient as 293T for recombinant protein expression. Further, PC3 offers an advantage in that the level of expression of the protein can be regulated by simple addition of n-butanol to the culture medium.", "link"=>"http://www.mendeley.com/research/translational-upregulation-highlevel-protein-expression-plasmid-vectors-mtor-activation-via-differen", "reader_count"=>16, "reader_count_by_academic_status"=>{"Researcher"=>6, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>5, "Student > Master"=>1, "Other"=>1, "Student > Bachelor"=>1, "Lecturer"=>1}, "reader_count_by_user_role"=>{"Researcher"=>6, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>5, "Student > Master"=>1, "Other"=>1, "Student > Bachelor"=>1, "Lecturer"=>1}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>3, "Agricultural and Biological Sciences"=>9, "Medicine and Dentistry"=>3, "Immunology and Microbiology"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>3}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>9}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>3}}, "group_count"=>0}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/809633"], "description"=>"<p>(<b>A</b>) PDGF-B mRNA levels were determined by RNase protection Assay. The 144 nt protected band corresponds to PDGF-B and the 120 nt band represents that of β-Actin mRNA. (<b>B</b>) RT-PCR of β-Gal, GFP and β-Actin mRNA in pcDNA3-β-Gal and pcDNA3-GFP transfected cells. (<b>C</b>) Radioimmunoprecipitation of PDGF-B protein expressed in pCMV-PDGF-B transfected cells using an N-terminal antibody <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014408#pone.0014408-Rao2\" target=\"_blank\">[69]</a>. (<b>D</b>) Levels of β-Gal, GFP and β-Tubulin proteins in pcDNA3- β-Gal and -GFP transfected cells. 50 µg of transfected cell lysate was analyzed for GFP and β-Gal levels by SDS-PAGE. (<b>E</b>) β-galactosidase assay using the β-Gal ELISA Kit from Roche Diagnostics. (<b>F</b>) Fluorescence microscopy and bright field (BF) images of 293T, PC3BM, HeLa and PC3 cells transfected with pcDNA-GFP reporter gene construct. (<b>G</b>) Analysis of the fold differences in expression of the reporter mRNA and protein levels between PC3 and HeLa, and PC3 and PC3BM.</p>", "links"=>[], "tags"=>["rna", "levels", "derived", "transfected", "vectors", "gfp", "293t", "ma104"], "article_id"=>479992, "categories"=>["Biotechnology", "Biochemistry", "Molecular Biology"], "users"=>["Prashanthi Karyala", "Nima D. Namsa", "Durga Rao Chilakalapudi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014408.g001", "stats"=>{"downloads"=>0, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Analysis_of_RNA_and_protein_levels_derived_from_the_transfected_expression_vectors_of_PDGF_B_GFP_and_946_Gal_in_PC3_PC3BM_HeLa_293T_and_MA104_cells_/479992", "title"=>"Analysis of RNA and protein levels derived from the transfected expression vectors of PDGF-B, GFP and β-Gal in PC3, PC3BM, HeLa, 293T and MA104 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 21:34:37"}
  • {"files"=>["https://ndownloader.figshare.com/files/809730"], "description"=>"<p>Analysis of total protein and/or phosphorylated forms of (<b>A</b>) mTOR and TSC2, (<b>B</b>) eIF4E, (<b>C</b>) 4EBP1, (<b>D</b>) S6, (<b>E</b>) eEF2 and eEF2K (<b>F</b>) p70S6K, (<b>G</b>) PI3K and PDK1, (<b>H</b>) Akt and (<b>I</b>) ERK1/2, p38 MAPK and PKC. 50 µg of cell lysate was used for analysis of mTOR, TSC2, PI3K, PDK1, Akt, eEF2, 4EBP1 and S6 and 100 µg was used for detection of phosphorylated forms and other proteins.</p>", "links"=>[], "tags"=>["blot", "proteins", "mtor"], "article_id"=>480096, "categories"=>["Biotechnology", "Biochemistry", "Molecular Biology"], "users"=>["Prashanthi Karyala", "Nima D. Namsa", "Durga Rao Chilakalapudi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014408.g002", "stats"=>{"downloads"=>2, "page_views"=>17, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Western_blot_analysis_of_key_target_proteins_of_mTOR_pathway_/480096", "title"=>"Western blot analysis of key target proteins of mTOR pathway.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 21:35:13"}
  • {"files"=>["https://ndownloader.figshare.com/files/809830"], "description"=>"<p>(<b>A</b>) Analysis of mTOR target proteins 4EBP1, S6, eEF2K and p70S6K. (<b>B</b>) mTOR and TSC2. (<b>C</b>) PI3K/Akt and ERK1/2. (<b>D</b>) PKC. 200 µg of cell lysate was used for PKC analysis and 50 µg was used for analysis of other proteins.</p>", "links"=>[], "tags"=>["translational", "proteins", "signaling", "pathway", "pc3", "293"], "article_id"=>480194, "categories"=>["Biotechnology", "Biochemistry", "Molecular Biology"], "users"=>["Prashanthi Karyala", "Nima D. Namsa", "Durga Rao Chilakalapudi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014408.g003", "stats"=>{"downloads"=>1, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Comparative_analysis_of_the_translational_regulatory_proteins_and_different_signaling_pathway_proteins_in_PC3_and_293_T_cells_/480194", "title"=>"Comparative analysis of the translational regulatory proteins and different signaling pathway proteins in PC3 and 293 T cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 21:35:46"}
  • {"files"=>["https://ndownloader.figshare.com/files/809985"], "description"=>"<p>Schematic representation of activation of mTOR by common and distinct signaling pathways leading to translational up-regulation in (A) PC3 and (B) 293T cell lines.</p>", "links"=>[], "tags"=>["activation", "mtor", "signaling", "pathways", "translational", "up-regulation", "pc3", "293t"], "article_id"=>480348, "categories"=>["Biotechnology", "Biochemistry", "Molecular Biology"], "users"=>["Prashanthi Karyala", "Nima D. Namsa", "Durga Rao Chilakalapudi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014408.g005", "stats"=>{"downloads"=>1, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Schematic_representation_of_activation_of_mTOR_by_common_and_distinct_signaling_pathways_leading_to_translational_up_regulation_in_A_PC3_and_B_293T_cell_lines_/480348", "title"=>"Schematic representation of activation of mTOR by common and distinct signaling pathways leading to translational up-regulation in (A) PC3 and (B) 293T cell lines.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 21:36:33"}
  • {"files"=>["https://ndownloader.figshare.com/files/809907"], "description"=>"<p>Effect of n-butanol on expression of (<b>A</b>) GFP; (<b>B</b>) β-Gal, and (<b>C</b>) Inhibition of phosphorylation of mTOR by n-butanol; Effect of U0126 (U0), Wortmannin (Wort) and LY294002 (LY) on (<b>D</b>) β-Gal and (<b>E</b>) GFP expression. Proteins were separated by 12–14% SDS-PAGE and detected by immunoblotting using antibodies specific to GFP and β-Gal.</p>", "links"=>[], "tags"=>["differential", "wortmannin", "ly294002", "phosphorylation", "mtor", "transfected", "pc3", "293t"], "article_id"=>480270, "categories"=>["Biotechnology", "Biochemistry", "Molecular Biology"], "users"=>["Prashanthi Karyala", "Nima D. Namsa", "Durga Rao Chilakalapudi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0014408.g004", "stats"=>{"downloads"=>3, "page_views"=>75, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Analysis_of_the_differential_effect_of_n_Butanol_U0126_Wortmannin_and_LY294002_on_reporter_gene_expression_and_phosphorylation_of_mTOR_in_transfected_PC3_and_293T_cells_/480270", "title"=>"Analysis of the differential effect of n-Butanol, U0126, Wortmannin and LY294002 on reporter gene expression and phosphorylation of mTOR in transfected PC3 and 293T cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 21:36:08"}

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Relative Metric

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