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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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HeLa cells: overexpression of importin alpha2

ABSTRACT: To test whether nuclear importin alpha2 can regulate transcription, we sought to examine gene expression changes in cells with nuclear accumulation of importin alpha2 by performing microarray analysis. We designed an experiment in which EGFP-fused full-length importin alpha2 was transfected into HeLa cells. To exclude the possibility that exogenous, full-length importin alpha2 protein enhances nuclear transport of karyophilic proteins such as transcription factors and thereby directly influences gene expression, a mutant of importin alpha2 which is mutated in the C-terminal CAS-binding domain was also transfected, so that it is never recycled to the cytoplasm and shows complete nuclear localization. We undertook to investigate whether there were changes in gene expression common to cells expressing the full-length importin alpha2 and the C-terminal mutant (C-mutant) isoform. EGFP vs. EGFP-importin alpha2 full length, or EGFP vs EGFP-importin alpha2 CAS-binding mutant. One replicate each. EGFP-expressing cells are used as a control.

ORGANISM(S): Homo sapiens

SUBMITTER: Yoichi Miyamoto

PROVIDER: E-GEOD-25303 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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HeLa cells: overexpression of importin alpha2

Project description:To test whether nuclear importin alpha2 can regulate transcription, we sought to examine gene expression changes in cells with nuclear accumulation of importin alpha2 by performing microarray analysis. We designed an experiment in which EGFP-fused full-length importin alpha2 was transfected into HeLa cells. To exclude the possibility that exogenous, full-length importin alpha2 protein enhances nuclear transport of karyophilic proteins such as transcription factors and thereby directly influences gene expression, a mutant of importin alpha2 which is mutated in the C-terminal CAS-binding domain was also transfected, so that it is never recycled to the cytoplasm and shows complete nuclear localization. We undertook to investigate whether there were changes in gene expression common to cells expressing the full-length importin alpha2 and the C-terminal mutant (C-mutant) isoform.

2010-11-24 | GSE25303 | GEO

Homo sapiens

Project description:HeLa cells: overexpression of importin alpha2

| PRJNA134789 | ENA

Expression data from HeLa cells treated with Casiopeina Cas-II-gly

Project description:Copper-based chemotherapeutic compounds Casiopeinas, have been presented as able to promote selective programmed cell death in cancer cells, thus being proper candidates for targeted cancer therapy. DNA fragmentation and apoptosis -in a process mediated by reactive oxygen species- for a number of tumor cells, have been argued to be the main mechanisms. However, a detailed functional mechanism (a model) is still to be defined and interrogated for a wide variety of cellular conditions; before establishing settings and parameters needed for their wide clinical application. Microarrays were used to determine the expression profile from HeLa cells in order to propose a model for the role played by intrinsic apoptosis triggered by the oxidative stress caused by Cas-II-gly. The cervix-uterine cell line HeLa was maintained at 37C in 5% CO2 under sterile conditions in Dulbecco's modified Eagle medium (DMEM, Sigma), supplemented with 10% fetal bovine serum (Sigma). Cells were treated with Cas II-gly in 96-well microplates by 6 h. HeLa cells whole genome gene expression experiments (triplicates for cases/controls) were performed in total mRNA extracted under the GPL570 protocol.

2012-10-31 | E-GEOD-41827 | biostudies-arrayexpress

New insights on the role of HCN in root hair elongation through single cell proteomics.

Project description:In order to identify the possible protein targets of cyanide in the regulation of root hair, we have carried out a single cell proteomic approach to characterize the protein atlas in wild type root hair cells compared to the cas-c1 mutant cells. Root hair specific cells were obtained from isolated protoplast from root tissues of Arabidopsis wt-pCOBL9:GFP and cas-c1-pCOBL9:GFP lines. To analyze the effect of the CAS-C1 mutation, we isolated 1 x 106 root hair cells by Fluorescence-activated cell sorting (FACS) from three independent replicate of wt-pCOBL9:GFP and cas-c1-pCOBL9:GFP roots. The extracted proteins from each sample were trypsin-digested and the digested peptides were analyzed by liquid chromatography-high resolution mass spectrometry (LC-MS/MS) for protein identification. In wild type samples, we have identified 3829 unique proteins at a false discovery rate below 1% (FDR<1%), that represent almost 10% of the total Arabidopsis proteome. In protein extract of the cas-c1-pCOBL9:GFP roots samples, we have identified 3972 proteins of which 3515 were commons in both cell types, 310 were only identified y wild type and 457 only in cas-c1 mutant (Fig. 5).

2024-01-26 | PXD028991 | Pride

Extensive identification and in-depth validation of importin 13 cargoes

Project description:Importin 13 is a bidirectional transport receptor that mediates both nuclear import and export of proteins. To expand the range of human importin 13 cargoes, we performed importin 13 binding experiments in combination with quantitative stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry. Apart from known importin 13 interaction partners, such as Ubc9 and eIF1A, more than 300 potential importin 13 cargoes were identified, several of which could be validated using cellular and biochemical assays.

2018-04-20 | PXD008459 | Pride

SILAC-Tp (12 importins) - Extensive cargo identification reveals distinct biological roles of the 12 importin pathways

Project description:Importin-beta family proteins are nucleocytoplasmic transport receptors (NTRs), and they transport most nuclear proteins as their cargoes into and out of the nuclei. Human cells have 20 species of importin-beta family NTRs, of which 10 (importin-beta, transportin-1, -2, -SR, importin-4, -5, -7, -8, -9, and -11) are nuclear import receptors and 2 (importin-13, and exportin-4) are bi-directional receptors. Here, we identified the import cargo proteins of these 12 NTRs by a method called SILAC-Tp, which employs stable isotope labeling with amino acids in cell culture (SILAC), an in vitro reconstituted transport system, and LC-MS/MS.

2017-01-20 | PXD004655 | Pride

RNAseq data for FACS sorted glial cells KPNA4 KO mice for Importin α4 deficiency induces psychiatric disorder-related behavioral deficits and neuroinflammation in mice.

Project description:Importin α4, which is encoded by the Kpna4 gene, is a well characterized nuclear-cytoplasmic transport factor known to mediate transport of transcription factors including NF-κB. Here, we report that Kpna4 knock-out (KO) mice exhibit psychiatric disorder-related behavioral abnormalities such as anxiety-related behaviors, deceased social interaction and sensorimotor gating deficits. Contrary to a previous study predicting attenuated NF-κB activity as a result of Kpna4 deficiency, we observed a significant increase in expression levels of NF-κB genes and pro-inflammatory cytokines such as TNFα, Il1β or Il-6 in the Prefrontal Cortex or Basolateral Amygdala of the KO mice. Moreover, examination of inflammatory responses in primary cells revealed that Kpna4 deficient cells have an increased inflammatory response, which was rescued by addition of not only full-length, but also a nuclear transport deficient truncation mutant of importin α4, suggesting contribution of its non-transport functions. Furthermore, RNAseq of sorted adult Microglia and Astrocytes and subsequent transcription factor analysis suggested increases in Polycomb repressor complex 2 (PRC2) activity in Kpna4 KO cells. Taken together, importin α4 deficiency induces psychiatric disorder-related behavioral deficits in mice, along with an increased inflammatory response and possible alteration of PRC2 activity in glial cells.

2024-04-18 | GSE264180 | GEO

Effect of Protease-resistant PML-RARα on the leukemogenic potential in a mouse model of Acute Promyelocytic Leukemia

Project description:Previous studies in our laboratory demonstrated that the azurophil granule protease neutrophil elastase (NE) cleaves PML-RARA (PR), the fusion protein that initiates acute promyelocytic leukemia (APL). Further, NE deficiency reduces the penetrance of APL in a murine model of this disease. We therefore predicted that NE-mediated PR cleavage might be important for its ability to initiate APL. To test this hypothesis, we generated a mouse expressing NE-resistant PR. These mice developed APL indistinguishable from wild type PR, but with significantly reduced latency (median leukemia-free survival of 274 days versus 473 days for wild type PR, p<0.001). Resistance to proteolysis may increase the abundance of full length PR protein in early myeloid cells, and our previous data suggested that non-cleaved PR may be less toxic to early myeloid cells. Together, these effects appear to increase the leukemogenicity of NE-resistant PR, contrary to our previous prediction. We conclude that NE deficiency may reduce APL penetrance via indirect mechanisms that are still NE dependent. Keywords: Time course U937 cells were transfected with one of three tagged constructs GFP,GFP-PR,GFP-PR-2VR sorted for GFP positivity at the indicated time points and harvested for RNA.

2010-09-01 | E-GEOD-21550 | biostudies-arrayexpress

Cells Comprising the Prostate Cancer Microenvironment Lack Recurrent Clonal Somatic Genomic Aberrations

Project description:Prostate cancer-associated stroma (CAS) plays an active role in malignant transformation, tumor progression, and metastasis. Molecular analyses of CAS have demonstrated significant changes in gene expression; however, conflicting evidence exists on whether genomic alterations in benign cells comprising the tumor microenvironment (TME) underlie gene expression changes and oncogenic phenotypes. This study evaluates the nuclear and mitochondrial DNA integrity of prostate carcinoma cells, CAS, matched benign epithelium and benign epithelium-associated stroma by whole genome copy number analyses, targeted sequencing of TP53, and fluorescence in situ hybridization. Comparative genomic hybridization (aCGH) of CAS revealed a copy-neutral diploid genome with only rare and small somatic copy number aberrations (SCNAs). In contrast, several expected recurrent SCNAs were evident in the adjacent prostate carcinoma cells, including gains at 3q, 7p, and 8q, and losses at 8p and 10q. No somatic TP53 mutations were observed in CAS. Mitochondrial DNA (mtDNA) extracted from carcinoma cells and stroma identified 23 somatic mtDNA mutations in neoplastic epithelial cells but only one mutation in stroma. Finally, genomic analyses identified no SCNAs, no loss of heterozygosity (LOH) or copy-neutral LOH in cultured cancer-associated fibroblasts (CAFs), which are known to promote prostate cancer progression in vivo. Agilent whole human genome aCGH oligonucleotide microarrays were used to assess copy number aberrations in 79 specimens from 20 patients as well as two pairs of cancer-associated fibroblasts (CAF) and benign/normal associated fibroblasts (NAF). 3 CAF/NAF sample pairs were also assessed for DNA copy number aberrations and copy-neutral LOH using the Infinium HumanOmniExpressExome BeadChip Kit.

2016-02-12 | E-GEOD-76456 | biostudies-arrayexpress

Plasmin activity promotes amyloid deposition in a new transgenic model of human transthyretin amyloidosis.

Project description:We have developed a new mouse model of transthyretin (TTR) amyloidosis using transgenic mice expressing the most fibrillogenic variant of TTR (S52P). Following seeding with amyloid fibrils, TTR amyloid is deposited mainly in the heart and tongue. The fibrils contain both full length and truncated (49-127) TTR. The presence of S52P TTR was confirmed by proteomics. Knockout of alpha2-antiplasmin enhanced amyloid deposition.

2021-12-27 | PXD027747 | Pride

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