Investigation Title RNAi knock down of VCP in human HeLa cells Comment[Submitted Name] VCP knockdown Experimental Design replicate_design reference_design cellular_modification_design dye_swap_design RNAi profiling by array Experimental Design Term Source REF mo mo mo EFO Comment[ArrayExpressReleaseDate] 2006-08-23 Comment[AEMIAMESCORE] 3 Comment[ArrayExpressAccession] E-MEXP-817 Comment[MAGETAB TimeStamp_Version] 2010-08-11 16:19:07 Last Changed Rev: 13058 Experimental Factor Name RNAi Experimental Factor Type RNAi Experimental Factor Term Source REF Person Last Name Rowicka DeMartino Adams Kujawa Kudlicki Nowis Wojcik Person First Name Maga George George Marek Andrzej Dominika Cezary Person Mid Initials N Person Email maga@work.swmed.edu Person Phone 2146456366 Person Fax Person Address 5323 Harry Hines Blvd Person Affiliation Biochemistry, University of Texas Southwestern Medical Center University of Texas Southwestern Medical Center University of Texas Southwestern Medical Center Medical University of Warsaw, Poland University of Texas Southwestern Medical Center Indiana School of Medicine Indiana School of Medicine Person Roles submitter Person Roles Term Source REF Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2006-08-23 PubMed ID 16914519 Publication DOI 16914519 Publication Author List Wojcik, Cezary; Rowicka, Maga; Kudlicki, Andrzej; Nowis, Dominika; McConnell, Elizabeth; Kujawa, Marek; DeMartino, George N. Publication Title Valosin-containing Protein (p97) Is a Regulator of Endoplasmic Reticulum Stress and of the Degradation of N-End Rule and Ubiquitin-Fusion Degradation Pathway Substrates in Mammalian Cells Publication Status journal_article Publication Status Term Source REF Experiment Description Study of the effects of the VCP knockdown. VCP (p97, yeast cdc48) is a hexameric AAA ATPase involved in various cellular functions including degradation of proteins by the ubiquitin-proteasome system. We examine the consequences of the reduction of VCP levels after RNAi of VCP in HeLa cells. We find ~30 transcripts upregulated in a sequence independent manner. Those transcripts encode proteins involved in endoplasmic reticulum stress, apoptosis, and amino acid starvation. Protocol Name P-MEXP-30646 P-MEXP-30645 P-MEXP-30004 P-MEXP-30608 P-MEXP-30609 P-MEXP-30006 P-MEXP-30610 Protocol Type specified_biomaterial_action grow nucleic_acid_extraction labeling labeling hybridization feature_extraction Protocol Description Small interfering RNAs (siRNAs) were obtained by chemical synthesis using 2'-ACE chemistry (Hartsel et al., 2005) from Dharmacon (Lafayette, CO). siRNAs were 2' deprotected, desalted, purified by polyacrylamide gel electrophoresis and duplexed by the manufacturer. The mass of each siRNA was verified by MALDI-TOF. After shipment in a dry form the siRNAs were suspended in the 1x universal buffer (20 mM KCl, 6 mM HEPES-KOH pH 7.5, and 0.2 mM MgCl2) at a 20 μM concentration, aliquoted and frozen at -20 C for further use. Two siRNAs were obtained targeting VCP and one siRNA targeting EGFP, a protein not found in HeLa cells. The first siRNA (positions 599-619 of human VCP mRNA; accession number NM_007126) called VCP-1 has been used previously and it was originally selected from five different siRNAs based on its efficiency (Wojcik et al., 2004a;Wojcik et al., 2004b). The second siRNA targeting VCP (positions 480-500), called VCP-2, was designed using Dharmacon's web site siRNA design center (Reynolds et al., 2004). As control for non-specific effects of RNAi we have designed and used the siRNA targeting EGFP (positions 1101-1018 of CVU55763, preceded by AA). RNAi was performed by single Oligofectamine®-mediated transfection (Invitrogen, Carlsbad, CA) as described previously (Wojcik et al., 2004a;Wojcik et al., 2004b). Cells were collected 4 days after the transfection. HeLa cells mock transfected with EGFP siRNA served as a control. HeLa cells were grown in Advanced DMEM (Invitrogen, Carlsbad, CA) supplemented with Gluta-MAX®, antibiotic/antimycotic solution and 2% fetal bovine serum (Gemini Bioproducts, Woodland, CA). Plasmids used for transfection were sequenced using CEQ 2000XL DNA Analysis System (Beckman Coulter, Fullerton, CA). Transfection was carried on using Lipofectamine 2000® according to manufacturer's instructions (Invtrogen, Carlsbad, CA). Following transient transfection, HeLa cells were used for the production of stable cell lines by selection with geneticin (Invitrogen, Carlsbad, CA). All clones which expressed a given protein showed accumulation of that protein after inhibition of the proteasome (data not shown). One clone from each group (UbG76V GFP, Ub-R-GFP, δCD3 and α1AT) with the highest basal expression level was selected for the study. None of those clones differed from the non-transfected or mock-transfected controls in morphology, growth characteristics, or time- and dose-dependent sensitivity to proteasome inhibitors (data not shown). RNA was isolated using the modified method of Chomczynski (Chomczynski and Sacchi, 1987) from HeLa cells 72 h after transfection with two different siRNAs targeting VCP (VCP-2 and VCP-6) or which have been treated for 6 h with 10 μM MG132, 10 μg/ml tunicamycin and 5 μM brefeldin A (all from Calbiochem, La Jolla, CA). RT-PCR was performed with the OneStep kit (Qiagen, Valencia, CA) using the pairs of primers amplifying the genes of interest. The samples were fluorescently labeled with either Cy3-dCTP or Cy5-dCTP (Amersham, Piscataway, NJ). The labeled probes were mixed with preheated ASAP Hybridization Buffer (Perkin-Elmer), and hybridized to an oligo array according to the manufacturer's instruction. The samples were fluorescently labeled with either Cy3-dCTP or Cy5-dCTP (Amersham, Piscataway, NJ). The labeled probes were mixed with preheated ASAP Hybridization Buffer (Perkin-Elmer), and hybridized to an oligo array according to the manufacturer's instruction. The labeled probes were mixed with preheated ASAP Hybridization Buffer (Perkin-Elmer), and hybridized to an oligo array according to the manufacturer's instruction. The slides were washed with SSC buffer from low to high stringency, and scanned by GenePix scanner (Axon Instruments Inc., Union City, CA) at 532 nm (Cy3) and 635 nm (Cy5). The Cy3 and Cy5 scans for each slide were superimposed, and the fluorescent ratio for each spot was obtained. The slides were scanned by GenePix scanner at 532 nm (Cy3) and 635nm (Cy5). Results are in single .gpr file for each replicate Protocol Parameters Extracted product;Amplification; Label used;Amplification; Amplification;Label used; Chamber type;Quantity of label target used;temperature; Protocol Hardware GenePix 4000A [Axon Instruments] Protocol Software GenePix [Axon Instruments] Protocol Contact Protocol Term Source REF mo The MGED Ontology SDRF File E-MEXP-817.sdrf.txt Term Source Name ncbitax The MGED Ontology ArrayExpress mo EFO The MGED Ontology Term Source File http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version