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GSE39497
Homo sapiens
10 Downloadable Samples
Affymetrix Human Gene 1.0 ST Array (hugene10st)
Description
Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of human ADPGK, which encodes an ADP-dependent glucokinase (ADPGK), in tumour cell lines. The hypothesis tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two tumour cell lines (H460 and HCT116). All four lines had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 213% for the parental line to 6.40.8% (p=0.002) and 4.30.8% (p=0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no effect on glycolysis as measured by glucose consumption or lactate formation under oxic or anoxic conditions, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis.
Publication Title
Expression and role in glycolysis of human ADP-dependent glucokinase.
Sample Metadata Fields
Cell line
View Samples- Mus musculus
- 24 Downloadable Samples
Illumina HiSeq 2500
Description
Mediator is regarded a general co-activator of RNA-Polymerase II dependent transcription but not much is known about its function and regulation in mouse pluripotent embryonic stem cells (mESC). One means of controlling Mediator function is provided by binding of the Cdk8 module (Med12, Cdk8, Ccnc and Med13) to Mediator. Here we report that the Cdk8 module subunit Med12 operates together with PRC1 to silence developmental key genes in the pluripotent state. At the molecular level, PRC1 is required to assemble ncRNA containing Med12-Mediator complexes at promoters of repressed genes. In the course of cellular differentiation the H2A-ubiquitin binding protein Zrf1 abrogates PRC1-Med12 binding and facilitates the recruitment of Cdk8 into Mediator. Remodeling of the Mediator-associated protein complex converts Mediator into a transcriptional enhancer that mediates ncRNA-dependent activation of Polycomb target genes Overall design: RNAseq of pluripotent (control, shNMC, shRing1b, shMed12, shCdk8, shZrf1) and early differentiating (control, shNMC, shMed12, shCdk8, shZrf1) stem cells in triplicates. Control would be normal E14TG2A mESCs. shNMC refers to E14TG2A cells stably transfected with a short hairpin that has no mammalian targets (Non Mammalian Control). All the other samples are indeed stably transfected with short hairpins against the indicated genes.
Publication Title
Dual role of Med12 in PRC1-dependent gene repression and ncRNA-mediated transcriptional activation.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples- Homo sapiens
- 8 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
Using a mimic miR-200c was restored to an aggressive, Type 2 endometrial cancer cell line, Hec50
Publication Title
MicroRNA-200c mitigates invasiveness and restores sensitivity to microtubule-targeting chemotherapeutic agents.
Sample Metadata Fields
Specimen part, Cell line
View Samples- Homo sapiens
- 5 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
Idiopathic pulmonary fibrosis (IPF) is associated with the accumulation of collagen-secreting fibroblasts and myofibroblasts in the lung parenchyma. Many mechanisms contribute to their accumulation, including resistance to apoptosis. In previous work, we showed that exposure to the pro-inflammatory cytokines, TNF- and IFN- reverses fibroblast resistance to apoptosis. The goal of this study was to investigate the underlying mechanism. Based on an initial interrogation of the transcriptomes of unstimulated and TNF- and IFN--stimulated primary lung fibroblasts and the lung fibroblast cell line, MRC5, we show here that among Fas-signaling pathway molecules, Fas expression was increased ~6-fold in an NF-B and p38mapk-dependent fashion. Prevention of the increase in Fas expression using Fas siRNAs blocked the ability of TNF- and IFN- to sensitize fibroblasts to Fas ligation induced-apoptosis; while enforced adenovirus-mediated Fas overexpression was sufficient to overcome basal resistance to Fas-induced apoptosis. Examination of lung tissues from IPF patients revealed low to absent staining of Fas in fibroblastic cells of fibroblast foci. Collectively, these findings suggest that increased expression of Fas is necessary and sufficient to overcome the resistance of lung fibroblasts to Fas-induced apoptosis. They also suggest that approaches aimed at increasing Fas expression by lung fibroblasts and myofibroblasts may be therapeutically relevant.
Publication Title
Increased cell surface Fas expression is necessary and sufficient to sensitize lung fibroblasts to Fas ligation-induced apoptosis: implications for fibroblast accumulation in idiopathic pulmonary fibrosis.
Sample Metadata Fields
Specimen part, Treatment
View Samples- Homo sapiens
- 3 Downloadable Samples
- IlluminaHiSeq2000
Description
Micro (mi)RNAs are small non-coding RNAs with key regulatory functions. Recent advances in the field allowed researchers to identify their targets. However, much less is known regarding the regulation of miRNA themselves. The accumulation of these tiny regulators can be modulated at various levels during their biogenesis from the transcription of the primary transcript (pri-miRNA) to the stability of the mature miRNA. Here, we studied the importance of the pri-miRNA secondary structure for the regulation of mature miRNAs accumulation. To this end, we used the Kaposi’s sarcoma herpesvirus, which encodes a cluster of twelve pre-miRNAs. Using small RNA profiling and quantitative northern blot analysis, we measured the absolute amount of each mature miRNAs in different cellular context. We found that the difference in expression between the least and most expressed viral miRNA could be as high as 60-fold. Using high-throughput selective 2’-hydroxyl acylation analyzed by primer extension (hSHAPE), we then determined the secondary structure of the long primary transcript. We found that highly expressed miRNAs derived from optimally structured regions within the pri-miRNA. Finally, we confirmed the importance of the local structure by swapping stem-loops for highly and lowly expressed miRNAs, which resulted in a perturbed accumulation of the mature miRNA. Overall design: Examination of sRNA profiles in 3 independent B cell lines expressing KSHV miRNAs or infected with KSHV, without replicate
Publication Title
Importance of the RNA secondary structure for the relative accumulation of clustered viral microRNAs.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 10 Downloadable Samples
- IlluminaHiSeq2000
Description
In this study, we used a RNA-sequencing (RNA-seq) approach to analyze the whole transcriptomes of human primary Keratinocytes (KC) at undifferentiated stage and differentiated stage with and without FOXC1 knockdown. Each treatment condition have 2 or 3 replicates. 10 million reads were collected. A total of 8202 genes were designated as present (RPKM>5 in at least one sample). 635 genes were differentially expressed (FDR<0.01, P<0.000774201). FOXC1 knock-down significantly impaired keratinocytes differentiation process. Overall design: Proliferating foreskin normal human keratinocytes were seeded in 24 well-dishes and transfected with scrambled siRNA and FOXC1 siRNA duplexes. The following day, half of the wells were differentiated in vitro by increasing Ca2+ concentration in culture media from 0.06mM to 1.3 mM for 5 days. The other half wells of cells were cultured in 0.06mM CaCl2. Both undifferentiated and differentiated cells were harvested for total RNA extraction. RNA sequencing libraries were made using Illumina RNA sequencing library construction protocol. RNA libraries were sequenced by 100bp reads on Illumina Hi-seq 2000.
Publication Title
Forkhead Box C1 Regulates Human Primary Keratinocyte Terminal Differentiation.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 6 Downloadable Samples
- IlluminaHiSeq2000
Description
In this study, we used a RNA-sequencing (RNA-seq) approach to analyze the whole transcriptomes of human primary Keratinocytes (KC) at different differentiation stages. An average of 72.56 million reads were collected for each sample: 87.68% of the sequences could mapped to the human genome, and 66.70% of sequence mapped to known human genes. A total of 17,446 ± 220 genes were expressed during the course of differentiation. 1024 transcription factors (TF) and genes encoding TF binding proteins were detected during the course of differentiation were expressed during the course of KC differentiation. Overall design: Foreskin normal human keratinocytes were differentiated in vitro by increasing Ca2+ concentration in culture media from 0.06mM to 1.3 mM. Undifferentiated cells and cells under differentiation for 24 hrs, 48hrs, 72 hours, 96 hours and 120 hours were harvested for total RNA extraction. RNA sequencing libraries were made using eukaryotic long non-coding RNA sequencing library construction protocol. RNA libraries were deep sequenced by 100bp paired-reads on Illumina His-seq 2000.
Publication Title
Forkhead Box C1 Regulates Human Primary Keratinocyte Terminal Differentiation.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 6 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
The molecular role of iron in gene expression remains poorly characterized. Moreover, the alterations in global gene expression after iron chelation remains unclear and are important to assess for understanding the molecular pathology of iron-depletion and the biological effects of iron chelators. We assessed the effect on whole genome gene expression of two iron chelators (desferrioxamine and Dp44mT). These studies are important for understanding the molecular and cellular effects of iron-depletion.
Publication Title
Cellular iron depletion stimulates the JNK and p38 MAPK signaling transduction pathways, dissociation of ASK1-thioredoxin, and activation of ASK1.
Sample Metadata Fields
Cell line
View Samples- Mus musculus
- 24 Downloadable Samples
- Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
This SuperSeries is composed of the SubSeries listed below.
Publication Title
Cancer stemness in Wnt-driven mammary tumorigenesis.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 24 Downloadable Samples
- Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
The Wnt/beta-catenin signalling pathway plays a central role in mammary stem cell homeostasis and in breast cancer. We employed the CD29hiCD24+ cell surface antigens to identify a subpopulation of mammary CSCs from Apc1572T/+, a mouse model for metaplastic breast adenocarcinoma, a subtype of triple-negative breast cancer in man. The MaCSCs are capable of recapitulating tumorigenesis when transplanted at low multiplicities in vivo, and of forming self-renewing organoids in vitro. Expression profiling of the different subpopulations sorted from normal and neoplastic mammary tissues revealed that the normal stem cell compartment is more similar to tumor cells than to their own differentiated progenies. Accordingly, Wnt signaling was found to be activated in the subpopulation encompassing normal mammary stem cells, though to a lesser degree than in the tumor cells. By comparing normal with cancer mouse mammary compartments, we were able to derive a MaCSC-specific signature composed of human orthologous genes able to predict poor survival, relapse and distant metastasis in human breast cancer. Finally, upon intravenous injection, only MaCSCs among the different tumor cell subpopulations are able to form metastatic lesions in a broad spectrum of anatomical sites. Overall, our data indicate that constitutive Wnt signaling activation interferes with mammary stem cell homeostasis leading to metaplasia and basal-like adenocarcinomas.
Publication Title
Cancer stemness in Wnt-driven mammary tumorigenesis.
Sample Metadata Fields
No sample metadata fields
View Samples