Intrinsic abnormalities in transplanted eutopic endometrium are believed to contribute to the pathogenesis of pelvic endometriosis. Herein, we investigated transcriptomic differences in human endometrial stromal fibroblasts (hESF) from women with (hESFendo) versus without (hESFnon-endo) endometriosis, in response to activation of the PKA pathway with 8-Br-cAMP. hESFnon-endo (n=4) and hESFendo (mild endometriosis, n=4) were isolated from eutopic endometrium and treated +/- 0.5mM 8-Br-cAMP for 96 hours. Purified total RNA was subjected to microarray analysis using the whole genome Gene 1.0 ST Affymetrix platform. 733 genes were regulated in cAMP-treated hESFnon-endo versus 172 genes in hESFendo, suggesting a blunted response to cAMP/PKA pathway activation in women with disease. Real-time PCR and ELISA validated the decreased expression of decidualization markers in hESFendo compared to hESFnon-endo. In the absence of disease, 8-Br-cAMP down-regulated progression through the cell-cycle due to a decrease in Cyclin D1, cyclin-dependent kinase 6 and cell division cycle 2, and an increase in cyclin-dependent kinase inhibitor 1A. However, cell cycle components in hESFendo were not responsive to 8-Br-cAMP, resulting in persistence of a proliferative phenotype. hESFendo treated with 8-Br-cAMP exhibited altered expression of immune response, extracellular matrix, cytoskeleton, and apoptosis genes. Changes in phosphodiesterase expression and activity were not different among experimental groups. Thus, eutopic hESF with increased proliferative potential may seed the pelvic cavity via retrograde menstruation and promote establishment, survival, and proliferation of endometriosis lesions, independent of hydrolysis of cAMP and likely due to an inherent abnormality in the PKA pathway in the presence of disease.
The protein kinase A pathway-regulated transcriptome of endometrial stromal fibroblasts reveals compromised differentiation and persistent proliferative potential in endometriosis.
Specimen part, Disease, Treatment
View SamplesUsing neutrophils from a cohort of normal individuals, we generated transcriptomic profile of 4 individuals. Overall design: Here we generated gene expression profile of neutrophil cells of 4 normal individuals.This data was integreated with DNA methylation profiles of same individuals.
Genome-wide DNA methylation map of human neutrophils reveals widespread inter-individual epigenetic variation.
No sample metadata fields
View SamplesER:RAS-G12V expressing IMR90 cells were transduced with N1ICD-containing or control vectors before treatment with either 100nM 4-OHT or vehicle for 6 days leading to Notch-induced senescence (NIS), RAS-induced senescence (RIS) or combined Notch and Ras-induced senescence (RNIS). Overall design: IMR90 cells expressing a 4-hydroxytamoxifen (4-OHT) inducible estrogen receptor (ER)-coupled RAS-G12V (ER:RAS-G12V) were transduced with N1ICD-FLAG-containing (residues 1758-2556 of human NOTCH1, as per Capobianco et al, Mol Cell Biol, 1997) or control vector before treatment with either 100nM 4-OHT or vehicle for 6 days , leading to RAS-induced senescence (RIS), NOTCH-induced senescence (NIS) or combined Ras & NOTCH-induced senescence (RNIS). The total RNA was then analysed for transcriptional profiling using mRNA-sequencing. There were 6 (six) biological replicates for each experimental condition. Untreated, vector-transduced ER:RAS IMR90 cells were the control condition
NOTCH1 mediates a switch between two distinct secretomes during senescence.
No sample metadata fields
View SamplesER:RAS-G12V expressing IMR90 cells were treated with either 100nM 4-OHT for 6 days or 100uM Etoposide for 2 days, followed by further culture for 5 days, leading to RAS-induced senescence (RIS) and DNA-damage induced senescence (DDIS) respectively. Overall design: IMR90 cells expressing a 4-hydroxytamoxifen (4-OHT) inducible estrogen receptor (ER)-coupled RAS-G12V (ER:RAS-G12V) were treated with either 100nM 4-OHT for 6 days or 100uM Etoposide for 2 days, followed by further culture for 5 days, leading to RAS-induced senescence (RIS) and DNA-damage induced senescence (DDIS) respectively. The total RNA was then analysed for transcriptional profiling using mRNA-sequencing. There were 8 (eight) biological replicatesfor each of the experimental conditions. Untreated ER:RAS IMR90 cells were the control condition
NOTCH1 mediates a switch between two distinct secretomes during senescence.
No sample metadata fields
View SamplesStress granules are small RNA-protein granules that modify the translational landscape during cellular stress to promote survival. The RhoGTPase RhoA is implicated in the formation of RNA stress granules. Our data demonstrate that the cytokinetic proteins ECT2 and AurkB are localized to stress granules in human astrocytoma cells. AurkB and its downstream target histone-3 are phosphorylated during arsenite-induced stress. Chemical (AZD1152-HQPA) and siRNA inhibition of AurkB results in fewer and smaller stress granules when analyzed utilizing high throughput fluorescent based cellomics assays. RNA immunoprecipitation with the known stress granule aggregates TIAR and G3BP1 was performed on astrocytoma cells and subsequent analysis revealed that astrocytoma stress granules harbour unique mRNAs for various cellular pathways including cellular migration, metabolism, translation and transcriptional regulation. Human astrocytoma cell stress granules contain mRNA that are known to be involved in glioma signaling and the mTOR pathway. These data provide evidence that RNA stress granules are a novel form of epigenetic regulation in astrocytoma cells, which may be targetable by chemical inhibitors and enhance astrocytoma susceptiblity to conventional therapy such as radiation and chemotherapy.
Epithelial Cell Transforming 2 and Aurora Kinase B Modulate Formation of Stress Granule-Containing Transcripts from Diverse Cellular Pathways in Astrocytoma Cells.
Specimen part, Cell line
View SamplesThe goal of this study was to compare mRNA from mammary epithelial cells of 3 mammary-specific Nmi knockout FVB with corresponding wildtype control. This was performed to obtain clues to the signaling pathways that were impacted in the mammary epithelial cells upon knocking-out Nmi expression. Overall design: To determine how the loss of Nmi contributed to a hyper-proliferative phenotype during puberty and lactation, we performed global RNAseq analysis from enriched mammary epithelial organoids from lactation day1 (L1), the time when Nmi protein expression in normal mammary epithelium is at its highest level. We compared 2 groups with 3 mice/group. We used second and third mammary glands of each mouse. These glands were isolated from mice on the first day of lactation, minced and dissociated in digestion medium (HBSS containing collagenase I (Sigma Aldrich, St. Louis, MO) (1mg/mL) and Pronase (Sigma Aldrich) (0.1mg/mL)) for two hours at 37C with shaking. Epithelial organoids were washed in PBS and enriched by pulse centrifugation to 1500rpm at least three times before subsequent assays.
Conditional knockout of N-Myc and STAT interactor disrupts normal mammary development and enhances metastatic ability of mammary tumors.
Specimen part, Subject
View SamplesPurpose: study the role of MALT1 auto-proteolysis in T cell receptor mediated activation of NF-kB. Methods: Jurkat cells were generated that express wild type MALT1, the auto-cleavage deficient MALT1-R149A mutant, the catalytic inactive MALT1-C464A mutant or the R149A-C464A double mutant (RACA). Expression of endogenous MALT1 was inactivated using TALEN technology for the Jurkat cells expressing MALT1-R149A (JDM-RA) and MALT1-C464A (JDM-CA). Illumina HISeq 2000 deep sequencing was performed to determine the mRNA profiles for MALT1, JDM-RA, JDM-CA and RACA cells in unstimulated conditions or after treatment with 75ng/ml PMA and 150 ng/ml ionomycin for 3 or 18 hrs. Results: PMA ionomycin stimulation of the MALT1 auto-cleavage defective JDM-RA cells fails to activate NF-kB-dependent transcription like for the MALT1 catalytic inactive JDM-CA cells and the double RACA mutant cells. Conclusion: MALT1 autoproteolysis is essential for transcription of NF-kB target genes Overall design: mRNA profiles of Jurkat expressing MALT1, MALT1-R149A, MALT1-C464A and MALT1-R149A-C464A after 0, 3 and 18 hours of stimulation with PMA and Ionomycin were generated by deep sequencing, in duplicate, using Illumina HISeq 2000
MALT1 auto-proteolysis is essential for NF-κB-dependent gene transcription in activated lymphocytes.
No sample metadata fields
View SamplesAngiogenesis is essential for tissue development, wound healing and tissue perfusion, with its dysregulation linked-to tumorigenesis, rheumatoid arthritis and heart disease. Here we show pro-angiogenic stimuli couple to NADPH oxidase-dependent generation of oxidants that catalyse an activating intermolecular-disulphide between regulatory-RI subunits of protein kinase A (PKA), which stimulates PKA-dependent ERK signalling. This is crucial to blood vessel growth as 'redox-dead' Cys17Ser RI knock-in mice fully resistant to PKA disulphide-activation have deficient angiogenesis in models of hind limb ischaemia and tumour-implant growth. Disulphide-activation of PKA represents a new therapeutic target in diseases with aberrant angiogenesis.
Deficient angiogenesis in redox-dead Cys17Ser PKARIα knock-in mice.
Specimen part
View SamplesEvaluation of transcripts from soybean seed tissue during seed fill for a pair of near-isogenic lines contrasting in seed protein and oil and carrying an introgression at the linkage group I protein QTL region. ****[PLEXdb(http://www.plexdb.org) has submitted this series at GEO on behalf of the original contributor, Yung-Tsi Bolon. The equivalent experiment is GM11 at PLEXdb.]
Complementary genetic and genomic approaches help characterize the linkage group I seed protein QTL in soybean.
Specimen part
View SamplesHuman cytomegalovirus (HCMV) is an important pathogen with multiple immune evasion strategies, including virally facilitated degradation of host antiviral restriction factors. Here, we describe a multiplexed approach to discover proteins with innate immune function on the basis of active degradation by the proteasome or lysosome during early phase HCMV infection. Using three orthogonal proteomic/transcriptomic screens to quantify protein degradation, with high confidence we identified 35 proteins enriched in antiviral restriction factors. A final screen employed a comprehensive panel of viral mutants to predict viral genes that target >250 human proteins. This approach revealed Helicase-like Transcription Factor (HLTF), a DNA helicase important in DNA repair, potently inhibits early viral gene expression but is rapidly degraded during infection. The functionally unknown HCMV protein UL145 facilitates HLTF degradation by recruiting the Cullin4 E3 ligase complex. Our approach and data will enable further identifications of innate pathways targeted by HCMV and other viruses. Overall design: 9 samples comprising three sets of three replicates (0h, 24h, 72h)
High-Definition Analysis of Host Protein Stability during Human Cytomegalovirus Infection Reveals Antiviral Factors and Viral Evasion Mechanisms.
Specimen part, Subject, Time
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