Mouse IMCD3 cell lines knocked-down for VPS33B, VIPAR and PLOD3
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Paul Gissen
PROVIDER: E-GEOD-81376 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Mouse IMCD3 cell lines knocked-down for VPS33B, VIPAR and PLOD3 are used as models of ARC syndrome, an autosomal recessive disorder cause by mutations in VPS33B or VIPAR. Previous work has shown that ARC syndrome causes epithelial cell polarisation defectes and mislocalisation of membrane proteins, so polarised IMCD3 cells were used as an experimental model. Affymetrix arrays were used to characterise the changes to the transcriptome when protein levels of VPS33B, VIPAR and PLOD3 are reduced.
2016-05-13 | GSE81376 | GEO
Project description:In the past three years the role of inflammatory cytokines and chemokines in tumour promotion and progression has been intensively studied. The chemokine receptor CXCR4 and its ligand CXCL12 are commonly expressed in malignant cells from primary tumours, metastases and also in malignant cell lines. To investigate the biological significance of this receptor/ligand pair, we knocked-down CXCR4 expression in ovarian cancer cell line IGROV-1 using shRNA, and established stable cell lines. Using Affymetrix microarrays we compared in vitro gene expression in parental IGROV-1 and IGROV-Mock cells with two clones of IGROV-shCXCR4 cells. Gene Set Enrichment Analysis (GSEA) of those genes which were altered by RNA interference of CXCR4 revealed evidence for a cell autonomous signaling network involving CXCR4, TNF-a, IL6 and Notch pathways in ovarian cancer cells. Experiment Overall Design: The Affymetrix GeneChip Human Genome U133Plus 2.0 arrays were used to define gene expression profiles in each cell line.
2009-01-13 | E-GEOD-13763 | biostudies-arrayexpress
Project description:Rapid advances in genotyping and sequencing technology have dramatically accelerated the discovery of genes underlying human disease. Elucidating the function of such genes and understanding their role in pathogenesis, however, remains challenging. Here, we introduce a genomic strategy to functionally characterize such genes, and apply it to LRPPRC (leucine-rich PPR-motif containing), a poorly studied gene that is mutated in Leigh Syndrome, French Canadian type (LSFC). We utilize RNAi to engineer an allelic series of cellular models in which LRPPRC has been stably silenced to different levels of knockdown efficiency. Using expression profiling, we discovered a specific role for LRPPRC in the expression of all mitochondrial DNA (mtDNA)-encoded mRNAs, but not the rRNAs, without affecting nuclear genes encoding mitochondrial proteins. We designed seven shRNAs targeting the LRPPRC cDNA sequence to silence its expression in MCH58 immortalized human fibroblasts. The LRPPRC expression level in these cells ranged from 9% to 100%. We demonstrated that knockdown cells carried stable silencing of the target gene and associated biochemical phenotypes. Our goal was to engineer stable knockdown cells which recapitulate the LSFC disease phenotype and subject them to expression profiling using Affymetric microarrays to identify genesets and biochemical pathways that are altered.
2010-03-23 | E-GEOD-20847 | biostudies-arrayexpress
Project description:Transcriptional profiling of SAS cells transfected with pLKO.1-LYRIC shRNA-B expression vector (desinaged as B) and control SAS cells (transfected with pLKO.1 vector, designated as CTL). Goal was to determine the effects of LYRIC knockdown on global SAS cells gene expression. Two-condition experiment, SAS cells transfected with pLKO.1-LYRIC shRNA-B expression vector (desinaged as B) v.s. control SAS cells (transfected with pLKO.1 vector, designated as CTL). Biological replicates: 4 control replicates, 4 transfected replicates.
2016-02-28 | E-GEOD-44766 | biostudies-arrayexpress
Project description:The BTB-Kelch protein KLHL3 is a Cullin3-dependent E3 ligase that mediates the ubiquitin-dependent degradation of kinases WNK1-4 to control blood pressure and cell volume. A crystal structure of KLHL3 has defined its binding to an acidic degron motif containing a PXXP sequence that is strictly conserved in WNK1, WNK2 and WNK4. Mutations in the second proline abrograte the interaction causing the hypertension syndrome pseudohypoaldosteronism type II. WNK3 shows a diverged degron motif containing 4 amino acid substitutions that remove the PXXP motif raising questions as to the mechanism of its binding. To understand this atypical interaction, we determined the crystal structure of the KLHL3 Kelch domain in complex with a WNK3 peptide. The electron density enabled the complete 11-mer WNK-family degron motif to be traced for the first time revealing several conserved features not captured in previous work, including additional salt bridge and hydrogen bond interactions. Overall, the WNK3 peptide adopted a conserved binding pose except for a subtle shift to accommodate bulkier amino acid substitutions at the binding interface. At the centre, the second proline was substituted by WNK3 Thr541, providing a unique phosphorylatable residue among the WNK-family degrons. Fluorescence polarisation and structural modelling experiments revealed that its phosphorylation would abrogate the KLHL3 interaction similarly to hypertension-causing mutations. Together, these data reveal how the KLHL3 Kelch domain can accommodate the binding of multiple WNK isoforms and highlight a potential regulatory mechanism for the recruitment of WNK3.
2022-03-07 | PXD031606 | Pride
Project description:SF-1 (NR5A1) was overexpressed (Over) or knocked down with shRNA (shRNA) in NCI-H295R human adrenocortical tumor cells and differential global gene expression analysed 48 hours later using Affymetrix GeneChip Human Gene 1.0ST arrays. Over: 5 million cells were transfected (Amaxa Nucleofection) with 10 ug of a pIRES2-AcGPF1-Nuc construct co-expressing SF-1 cDNA and GFP. For experimental control, a mutagenized pIRES2 construct, bearing the G35E mutation in SF-1 that impairs its transactivation function in vivo and in vitro was used. shRNA: 5 million cells were transfected (Amaxa Nucleofection) with 10 ug of the SureSilencing shRNA Plasmid for Human NR5A1 with GFP marker kit (SABioscience). For experimental control, mismatch constructs provided in the kit were used. In both experiments (Over and shRNA), cells were harvested, prepared, and submitted to fluorescence-activated cell sorting (FACS) in a MoFlo XDP sorter 48 hours after transfection. Viable GFP-expressing cells were pooled and resuspended in TRIzol reagent for RNA extraction. Total RNA was extracted, and RNA quality control performed using a 2100 Bioanalyzer. RNA samples were processed using the Affymetrix GeneChip WT Sense Target Labeling kit, starting with 200 ng total RNA. Four independent Over experiments and five independent shRNA experiments were performed and samples of labeled fragmented cDNA were hybridized to Affymetrix GeneChip Human Gene 1.0 ST Arrays. Based on quality control of array data (R/Bioconductor and Partek Genomics Suite), two Over arrays (one SF-1 wild type and one mutant control) and one shRNA array (mismatch control) were excluded and are not deposited here. Differential gene expression analysis was performed using the limma package in R/Bioconductor. A Benjamini-Hochberg-corrected P value cut-off of 0.05 was used to select significant differentially expressed genes in each setting (Over and shRNA). Finally, results from both analyses were combined to identify a subset of positively-regulated SF-1 target genes in these cells, ie transcripts up-regulated by SF-1 overexpression (Over) and down-regulated by SF-1 knockdown (shRNA). Four SF-1 overexpression experiments (Over; intervention = overexpression of wild type SF-1; control = overexpression of functionally-impaired mutant G35E SF-1) and five SF-1 knockdown experiments (shRNA; intervention = SF-1-specific small hairpin RNA; control = shRNA mismatch control) were performed in NCI-H295R cells. Differential gene expression in each setting was analysed using GeneChip Human Gene 1.0ST arrays. Data for arrays that met quality control are presented here: Over, 3 intervention and 3 control arrays; shRNA, 5 intervention and 4 control arrays. Total of 15 arrays.
2011-01-11 | E-GEOD-26529 | biostudies-arrayexpress
Project description:Integrin trafficking to and from membrane adhesions is a crucial mechanism that dictates many aspects of a cell’s behaviour, including motility, polarisation, and invasion. In endothelial cells (ECs), the intracellular traffic of α5-integrin is regulated by both neuropilin 1 (NRP1) and neuropilin 2 (NRP2), yet the redundancies in function between these co-receptors remain unclear. Moreover, the endocytic complexes that participate in NRP-directed-traffic remain poorly annotated. Here we identify an important role for the GTPase-activating protein p120RasGAP in ECs, promoting the recycling of α5-integrin from early endosomes. Mechanistically, p120RasGAP enables transit of endocytosed α5-integrin-NRP1-NRP2 complexes to Rab11+ recycling endosomes, promoting cell polarisation and fibronectin (FN) fibrillogenesis. Silencing of both NRP receptors, or p120RasGAP, resulted in the accumulation of α5-integrin in early endosomes, a loss of α5-integrin from surface adhesions, and attenuated EC polarisation. Endothelial-specific deletion of both NRP1 and NRP2 in the postnatal retina recapitulated our in vitro findings, severely impairing FN fibrillogenesis and polarised sprouting. Our data assign an essential role for p120RasGAP during integrin traffic in ECs and support a hypothesis that NRP receptors co-traffic internalised cargoes. Importantly, we utilise comparative proteomics analyses to, for the first time, isolate a comprehensive map of NRP1-dependent and NRP2-dependent-α5-integrin interactions in ECs.
2024-05-20 | PXD051778 | Pride
Project description:We analysed whole PolyA+ RNA from human osteosarcoma U2OS cells depleted for human Cactin or transfected with a control shRNA. Two independent shRNAs targeting human Cactin (shCac_C and shCac_D), a control shRNA (shCtrl), a single cell line (U2OS)
2016-06-01 | E-GEOD-82070 | biostudies-arrayexpress
Project description:Identifying PDEF regulated genes may shed light on the mechanism by which PDEF may induce breast cancer progression. To that purpose, we have used the MCF-7 human breast tumor cell line model to identify PDEF induced genes. Briefly, PDEF expression was down regulated by shRNA in MCF-7 cells and RNA probes from PDEF-down regulated and control MCF-7 cells were used to screen the Affymetrics HG-U133A Gene Chips. This analysis found 62 genes that were induced 2-fold or higher by PDEF. Further analysis of 3 of these genes namely S100A7, CEACAM6 and B7-H4 in primary breast tumors showed CEACAM6 as a frequently elevated and co-exressed gene with PDEF in these tumors. We previously reported a role for PDEF (prostate derived Ets transcription factor) in breast tumor progression and its association with poor clinical outcome in ER+ breast cancer. To gain further insights into PDEF action in breast cancer, we down regulated PDEF expression by shRNA in MCF-7 human breast tumor cell line, and screened the HG-U133A human gene chips with probes from PDEF down-regulated and control MCF-7 cells. This analysis identified CEACAM6 as one of the genes induced by PDEF. Further analysis of CEACAM6 expression in relation to PDEF in 93 ER+ primary breast tumors showed largely concordant expression of these molecules. To our knowledge, our findings of CEACAM6 as a PDEF induced gene and their elevated co-expression in breast cancer have not been described before. Data from one replicate experiment is included as a representative example of the data obtained. HG-U133A gene chip pairs were screeened with biotinylated RNA probes from PDEF-down regulated MCF-7 cells (experimental) or from control MCF-7 cells.
2012-04-30 | E-GEOD-37662 | biostudies-arrayexpress
Project description:To identify early processes in carcinogenesis, we used an in vitro model, based on the initiating event in cervical cancer, human papillomavirus (HPV) transformation of keratinocytes. We compared gene expression in primary keratinocytes (K) and HPV16-transformed keratinocytes from early (E) and late (L) passages, and from benzo[a]pyrene treated L cells (BP). The transformed cells exhibit similar transcriptional changes to clinical cervical carcinoma. We revealed a contraction in expression of the apoptotic network during HF1 cell transformation, which affected the ability of L and BP cells to execute apoptosis, but did not lead to resistance to apoptotic stimuli. The contraction in the apoptotic machinery during the process of transformation was accompanied by a switch from apoptosis to necrosis in response to CDDP. The shrinkage of the pro- and anti-apoptotic networks appears to be part of a general contraction in the number of genes transcribed in L and BP cells. We also identified a large group of genes with induced expression, which are involved in cell metabolism and cell cycle, suggesting increased investment of the transformed cell in cellular proliferation. We hypothesize that the decrease in expression of many diverse pathways, including the pro- and anti-apoptotic networks, cuts the energy requirements for cell maintenance, allowing energy to be diverted towards rapid cell proliferation. This study supports the hypothesis that the process of cancer transformation may be accompanied by a shift from apoptosis to necrosis. Experiment Overall Design: We isolated mRNA from three independent cultures each of Keratinocytes and HPV16-immortalized keratinocytes : E, L and BP cells, grown under basal conditions. cDNA was prepared and hybridized to the Human Genome U133A Array (Affymetrix).
2009-05-02 | E-GEOD-15156 | biostudies-arrayexpress