Filters
Technology
Platform
E-MEXP-549
Homo sapiens
21 Downloadable Samples
Affymetrix Human Genome U133A Array (hgu133a)
Description
The purpose of the experiment was to generate a time course of gene expression following irradiation. The goal was then to model this data to extract hidden variables - chiefly, the activity profiles of the p53 transcription factor. Using this information the aim was to predict which transcripts changed by IR were targets of p53. Cells in log phase (1 x 106 ml-1) were ?-irradiated with 5 Gy at room temperature (RT) at a dose rate of 2.45 Gy per minute with a 137Cs ?-irradiator. Cells were harvested at 0, 2, 4, 6, 8, 10 and 12 hours, and RNA and protein were extracted (Trizol, Invitrogen). Affymetrix U133A arrays were hybridized as standard (www.affymetrix.co.uk). Array quality was determined using R and GCOS .rpt file values. The time course was replicated three times from independent cell preparations.
Publication Title
Ranked prediction of p53 targets using hidden variable dynamic modeling.
Sample Metadata Fields
Specimen part, Disease, Cell line, Time
View Samples- Homo sapiens
- 6 Downloadable Samples
Illumina HiSeq 2000
Description
Type 1 diabetes (T1D) is caused by autoimmune destruction of pancreatic ß cells. Mounting evidence supports a central role for ß-cell alterations in triggering the activation of self-reactive T-cells in T1D. However, the early deleterious events that occur in ß cells, underpinning islet autoimmunity are not known. We hypothesized that epigenetic modifications induced in ß cells by inflammatory mediators play a key role in initiating the autoimmune response. We analyzed DNA methylation (DNAm) patterns and gene expression in human islets exposed to IFNa, a cytokine associated with T1D development. We found that IFNa triggers DNA demethylation and increases expression of genes controlling inflammatory and immune pathways. We then demonstrated that DNA demethylation was caused by up-regulation of the exoribonuclease, PNPase Old-35 (PNPT1), which caused degradation of miR-26a. This in turn promoted the up-regulation of ten-eleven translocation TET2 enzyme and increased 5-hydoxymethylcytosine levels in human islets and pancreatic ß-cells. Moreover, we showed that specific IFNa expression in the ß cells of IFNa-INS1CreERT2 transgenic mice, led to development of T1D that was preceded by increased islet DNA hydroxymethylation through a PNPT1/TET2-dependent mechanism. Our results suggest a new mechanism through which IFNa regulates DNAm in ß cells, leading to changes in expression of genes in inflammatory and immune pathways that can initiate islet autoimmunity in T1D. Overall design: We exposed human pancreatic islets from three donors to 2000 IU IFNa and assessed gene expression by RNAseq. The cDNA library was prepared using Illumina TruSeq RNA Sample Prep Kits. Next generation sequencing was performed on Illumina HiSeq2000 using the Single-Read Cluster Generation kit v2 and SBS Sequencing kit v3. Image analysis and base calling were conducted using the SDS 2.5/RTA1.5 software.
Publication Title
Epigenetic modulation of β cells by interferon-α via PNPT1/mir-26a/TET2 triggers autoimmune diabetes.
Sample Metadata Fields
Specimen part, Disease stage, Treatment, Subject
View Samples- Homo sapiens
- 6 Downloadable Samples
Description
The exosome-independent exoribonuclease DIS3L2 is mutated in Perlman syndrome. Here we used extensive global transcriptomic and targeted biochemical analyses to identify novel DIS3L2 substrates in human cells. We show that DIS3L2 regulates pol II transcripts, comprising selected canonical and histone-coding mRNAs, and a novel FTL_short RNA from the ferritin mRNA 5'' UTR. Importantly, DIS3L2 contributes to surveillance of pre-snRNAs during their cytoplasmic maturation. Among pol III transcripts, DIS3L2 particularly targets vault and Y RNAs and an Alu-like element BC200 RNA, but not Alu repeats, which are removed by exosome-associated DIS3. Using 3'' RACE-Seq, we demonstrate that all novel DIS3L2 substrates are uridylated in vivo by TUT4/TUT7 poly(U) polymerases. Uridylation-dependent DIS3L2-mediated decay can be recapitulated in vitro, thus reinforcing the tight cooperation between DIS3L2 and TUTases. Together these results indicate that catalytically inactive DIS3L2, characteristic of Perlman syndrome, can lead to deregulation of its target RNAs to disturb transcriptome homeostasis. Overall design: To investigate DIS3L2 functions genome-wide, total RNA samples were collected from model cell lines producing either WT or mut DIS3L2 three days after induction with doxycycline. The RNA samples were rRNA-depleted before preparation of strand-specific total RNA libraries according to the standard TruSeq (Illumina) protocol. TruSeq library preparation favours RNA molecules longer than 200 nt, and shorter transcripts are suboptimal for sequencing via this protocol. Thus, to obtain information about potential DIS3L2 RNA substrates with lengths between 20 and 220 nt, another RNA-Seq was carried out in parallel (with size selection through gel purification). The stable inducible HEK293 cell lines producing DIS3L2 variants were obtained using “pAL_01” and “pAL_02” plasmid constructs and the Flp-In™ T-REx™ system according to the manufacturer’s guidelines. “pAL_01” and “pAL_02” plasmids are vectors for co-expression of recoded C-terminal FLAG-tagged DIS3L2 [wild type (WT) variant or its catalytic mutant counterpart (mut), respectively] and sh-miRNAs directed against endogenous DIS3L2 mRNA.
Publication Title
Perlman syndrome nuclease DIS3L2 controls cytoplasmic non-coding RNAs and provides surveillance pathway for maturing snRNAs.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 21 Downloadable Samples
- IlluminaHiSeq2000
Description
A multi-subunit exosome complex is a major eukaryotic exoribonuclease that in the cytoplasm requires the SKI complex for activity. In yeast, SKI forms a heterotetramer and delivers RNA substrates directly into the exosome channel. Such cooperation requires Ski7 protein, which links the exosome and SKI complexes. However, since the human genome does not encode an orthologue of the yeast Ski7, the factor mediating SKI and exosome linkage in human cells is unknown. Proteomic analysis revealed that the human cytoplasmic exosome interacts with HBS1LV3, a protein encoded by a newly discovered short splicing isoform of HBS1L. HBS1LV3 recruits the SKI complex to the exosome. In contrast, the canonical HBS1L variant, HBS1LV1, acting as a ribosome dissociation factor, does not associate with the exosome and instead interacts with the mRNA surveillance factor PELOTA. HBS1LV3 contains a new domain of unknown structure with the short linear motif RxxxFxxxL, which is responsible for exosome binding, and may interact with the exosome core subunit RRP43 in way that resembles the association between Rrp6 RNase and Rrp43 in yeast. Depletion of HBS1LV3 and the SKI complex helicase SKI2W similarly affected the transcriptome by strongly upregulating a large number of genes. Moreover, following HBS1LV3 or SKI2W depletion the half-lives of representative upregulated mRNAs were increased, thus supporting the involvement of HBS1LV3 and SKI2W in the same mRNA degradation pathway. In contrast, HBS1LV1 depletion had little effect on transcriptome homeostasis. Our data indicate that human HBS1LV3 is the long-sought factor that links the exosome and SKI complexes to regulate cytoplasmic mRNA decay. Overall design: Examination of siRNA-mediated silencing in HEK293 cell lines. To identify transcripts that are degraded by cytoplasmic SKI/HBS1LV3/exosome supercomplexes, we used specific siRNAs to knock down HBS1LV1, HBS1LV3 or SKIV2L gene expression in (i) WT HEK293 cells and (ii) HEK293 cells rescued with siRNA insensitive protein. Analyses were performed in triplicate.
Publication Title
A short splicing isoform of HBS1L links the cytoplasmic exosome and SKI complexes in humans.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 6 Downloadable Samples
- Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
Ingestion of collagen peptide elicits beneficial effects on the body. Improvement of blood lipid is one of the effects, but its mechanism remains unclear. Male BALB/cCrSlc mice were bred with the AIN-93M diet containing 14% casein or AIN-93M-based low-protein diet containing 10% casein or diet containing 6% casein+4% collagen peptide (n=12/group) for 10 weeksTotal, free, and esterified cholesterol levels in the blood decreased in the collagen peptide group. DNA microarray analysis of the liver revealed that expression of the genes related to lipid metabolic process, such as PPAR signaling pathway and fatty acid metabolism, increased in the collagen peptide group compared to the 10% casein group. In contrast, expression of the genes related to unfolded protein response (UPR) and protein level of phospho-IRE1 decreased. Our data suggest that lipid metabolism in the liver was altered by collagen ingestion, which probably results in the decreased levels of blood cholesterol.
Publication Title
Collagen peptide ingestion alters lipid metabolism-related gene expression and the unfolded protein response in mouse liver.
Sample Metadata Fields
Sex, Age, Specimen part
View Samples- Homo sapiens
- 34 Downloadable Samples
- IlluminaHiSeq2000
Description
Cell purification technology combined with whole transcriptome sequencing and small molecule agonist of hematopoietic stem cell self-renewal has allowed us to identify the endothelial protein c receptor protein (EPCR) as a surface maker that defines a rare subpopulation of human cells which is highly enriched for stem cell activity in vivo. EPCR-positive cells exhibit a robust multi-lineage differentiation potential and serial reconstitution in immunocompromised mice. In culture, most if not all of the HSC activity is detected in the EPCR+ subset, arguing for the stability of this marker on the surface of cultured cells, a feature not found with more recently described markers such as CD49f. Functionally EPCR is essential for human HSC activity in vivo. Cells engineered to express low EPCR expression proliferate normally in culture but lack the ability to confer long-term reconstitution. EPCR is thus a stable marker for human HSC. Its exploitation should open new possibilities in our effort to understand the molecular bases behind HSC self-renewal. Overall design: Examining 3 cellular subsets: EPCR+, EPCRlow, EPCR- derived form CD34+CD45RA- cord blood cells after 7 day expansion in UM171
Publication Title
EPCR expression marks UM171-expanded CD34<sup>+</sup> cord blood stem cells.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 53 Downloadable Samples
- IlluminaHiSeq2000
Description
RNASeq data for mPB or CB-derived CD34+ exposed to UM171 Overall design: human mobilized peripheral blood or cord blood-derived CD34(+) cells were cultured for 16 hours with vehicle (DMSO), dose response of UM171 [11.9nM, 19nM, 30.5nM, 48.8nM, 78.1nM and 125nM], SR1 [500nM] and combination of( UM171 [48.8nM]+SR1 [500nM])
Publication Title
UM171 induces a homeostatic inflammatory-detoxification response supporting human HSC self-renewal.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 12 Downloadable Samples
- Illumina HiSeq 2500
Description
Quiescent stem cells of glioblastoma (GBM), a malignant primary brain tumor, are potential sources for recurrence after therapy. However, the gene expression program underlying the physiology of GBM stem cells remains unclear. We have isolated quiescent GBM cells by engineering them with a knock-in H2B-GFP proliferation reporter and expanding them in a 3D tumor organoid model that mimics tumor heterogeneity. H2B-GFP label retaining quiescent cells were subjected to stem cell assays and RNA-Seq gene expression analysis. While quiescent GBM cells were similar in clonal culture assays to their proliferative counterparts, they displayed higher therapy resistance. Interestingly, quiescent GBM cells upregulated epithelial-mesenchymal transition (EMT) genes and genes of extracellular matrix components. Our findings connect quiescent GBM cells with an EMT-like shift, possibly explaining how GBM stem cells achieve high therapy resistance and invasiveness, and suggest new targets to abrogate GBM. Overall design: Glioblastoma cancer cells in 3D organoid culture were pulsed for 2 weeks with H2B-GFP, then chased either 2 or 4 weeks. Label-retaining GFP-high cells (quiescent) were separated from bulk population, and both populations were analyzed by RNA-Seq.
Publication Title
Gene signatures of quiescent glioblastoma cells reveal mesenchymal shift and interactions with niche microenvironment.
Sample Metadata Fields
Specimen part, Subject
View Samples- Mus musculus
- 9 Downloadable Samples
- Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
Analysis of bone marrow derived macrophages (BMDM) incubated with dexamethasone&IL4 (Dexa+IL4), B16F1 tumor conditioned medium (cmB16), and B16F1 tumor conditioned medium supplemented with dexamethasone&IL4 (cmB16+dexa+IL4). Results allow detection of genes that require synergistic stimulation of tumor factors and Th2 cytokines.
Publication Title
Synergistic activation by p38MAPK and glucocorticoid signaling mediates induction of M2-like tumor-associated macrophages expressing the novel CD20 homolog MS4A8A.
Sample Metadata Fields
Specimen part
View Samples- Mus musculus
- 6 Downloadable Samples
- Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
Tumor-associated macrophages (TAMs) represent alternatively activated (M2) macrophages that support tumor growth. Previously, we have described a special LYVE-1(+) M2 TAM subset in vitro and in vivo; gene profiling of this TAM subset identified MS4A8A as a novel TAM molecule expressed in vivo by TAM in mammary carcinoma and malignant melanoma. In vitro, Ms4a8a mRNA and MS4A8A protein expression was strongly induced in bone marrow-derived macrophages (BMDMs) by combining M2 mediators (IL-4, glucocorticoids) and tumor-conditioned media (TCM). Admixture of MS4A8A(+) TCM/IL-4/GC-treated BMDM significantly enhanced the tumor growth rate of subcutaneously transplanted TS/A mammary carcinomas. Upon forced overexpression of MS4A8A, Raw 264.7 macrophage-like cells displayed a special gene signature. Admixture of these MS4A8A(+) Raw 264.7 cells also significantly enhanced the tumor growth rate of subcutaneously transplanted mammary carcinomas. To identify the signaling pathways involved in synergistic induction of MS4A8A, the major signaling cascades with known functions in TAM were analyzed. Although inhibitors of NF-B activation and of the MAPK JNK and ERK did not show relevant effects, the p38/ MAPK inhibitor SB203580 strongly and highly significantly (p > 0.001) inhibited MS4A8A expression on mRNA and protein level. In addition, MS4A8A expression was restricted in M2 BMDM from mice with defective GC receptor (GR) dimerization indicating that classical GR gene regulation is mandatory for MS4A8A induction. In conclusion, expression of MS4A8A within the complex signal integration during macrophage immune responses may act to fine tune gene regulation. Furthermore, MS4A8A(+) TAM may serve as a novel cellular target for selective cancer therapy.
Publication Title
Synergistic activation by p38MAPK and glucocorticoid signaling mediates induction of M2-like tumor-associated macrophages expressing the novel CD20 homolog MS4A8A.
Sample Metadata Fields
Specimen part
View Samples