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SRP015449
Homo sapiens
5 Downloadable Samples
Description
Purpose: Development of resistance to tamoxifen is an important clinical issue in the treatment of patients with breast cancer. Tamoxifen resistance may be the result of the acquisition of epigenetic regulation such as DNA methylation within breast cancer cells resulting in changed mRNA expression of genes being pivotal for estrogen dependent growth. Alternatively, tamoxifen resistance may be due to selection of preexisting resistant cells, which may exhibit cancer stem-like characteristics or a combination of the two mechanisms. Methods: To evaluate the contribution of these possible mechanisms to tamoxifen resistance, we applied modified DNA methylation-specific digital karyotyping (MMSDK) and digital gene expression (DGE) in combination with massively parallel sequencing to analyze a well-established tamoxifen resistant cell line model: MCF-7/S0.5 (tamoxifen sensitive parental cell line) and 4 high-dosage tamoxifen selected resistant offspring sublines (MCF-7/TAMR-1, MCF-7/TAMR-4, MCF-7/TAMR-7 and MCF-7/TAMR-8). MMSDK uses BssHII as mapping enzyme (DNA methylation sensitive enzyme). Both MMSDK and DGE use NlaIII and MmeI to produce 20-21 bp tag. The indexed single-end sequencing was performed by Illumina HiSeq 2000 in BGI-Shenzhen. A dynamic programming algorithm-FASTX-Toolkit implemented in Perl was used to trim the adaptor sequence. The trimmed tags were subjected to quality filtering, so that only tags with sequencing quality higher than 30 for more than 80% of the nucleotides were used for subsequent analysis. For MMSDK tag mapping, we generated a simulated reference library, i.e., BssHII reference library, by in silico enzyme digestion of the human genome (hg19, UCSC) regardless of the methylation state. This library was used as reference for subsequent mapping of the tags in the MMSDK analysis. In the DGE analysis, refMrna (hg19, UCSC) was used as reference for mapping cDNA tags. Subsequently, the Burrows–Wheeler Aligner (BWA) procedure for aligning the MMSDK and DGE tags to the simulated BssHII reference library and refMrna reference library, respectively, was applied. Results: MMSDK libraries using BssHII/NlaIII were generated from the parental tamoxifen sensitive subline MCF-7/S0.5 and the 4 TAMR cell lines: TAMR-1, TAMR-4, TAMR-7 and TAMR-8. The 5 indexed MMSDK libraries were sequenced in one lane and 1.38 Gb clean tag data for all 5 cell lines were obtained, with an average sequencing amount of ~270 Mb per library. On average, 59.5 % of the tags with mapping quality = 20 were mapped back to the simulated BssHII/NlaIII reference library. DGE libraries were also generated from MCF-7/S0.5 and the 4 TAMR cell lines. The 5 indexed DGE libraries were sequenced in one lane and obtained 1.71 Gb clean tag data for all 5 cell lines with an average sequencing amount of ~340 Mb per library. On average, 40.8 % with mapping quality = 20 were mapped back to the simulated NlaIII human transcriptome (refMrna reference library). Our present study demonstrates large differences in global gene expression and DNA methylation profiles between parental tamoxifen-sensitive cell line and 4 high-dosage tamoxifen treatment selected resistant sublines. The tamoxifen resistant cell lines exhibited globally higher methylation level than the parental cell line and an inverse relationship between gene expression and DNA methylation in the promoter regions were noticed. High expression of SOX2 and alterations of other SOX gene family members, E2F gene family members and RB-related pocket protein genes as well as highlighted stem cell pathways imply that cancer initiating cells/stem cells are involved in the resistance to tamoxifen. Overall design: DNA methylation and mRNA expression profiles from tamoxifen sensitive parental cell line MCF-7/S0.5 and 4 high dosage of tamoxifen selected resistant offspring sublines (MCF-7/TAMR-1, MCF-7/TAMR-4, MCF-7/TAMR-7 and MCF-7/TAMR-8) were analyzed by MMSDK and DGE methods, respectively, in combination of massively parallel sequencing, using Illumina HiSeq 2000
Publication Title
Integrative analyses of gene expression and DNA methylation profiles in breast cancer cell line models of tamoxifen-resistance indicate a potential role of cells with stem-like properties.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 11 Downloadable Samples
Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
The most common oncogenic mutations in multiple myeloma (MM) affect N- and K-RAS leading to constitutive activation of RAS-dependent signaling. Signal transduction via RAS, Raf and MAPK has been well described as a canonical pathway. In accordance with this assumption, we showed that the activity of the MEK/ERK module is strictly dependent on pan-Raf activity. However, inhibition of MEK/ERK has no or only minor effects on MM cell survival, whereas oncogenic Ras and pan-Raf critically contribute to survival of multiple myeloma cells. Therefore, we aimed to learn more about Raf-dependent but MEK-independent signaling effectors.
Publication Title
Pan-Raf co-operates with PI3K-dependent signalling and critically contributes to myeloma cell survival independently of mutated RAS.
Sample Metadata Fields
Cell line
View Samples- Homo sapiens
- 36 Downloadable Samples
- Affymetrix Human Gene 1.0 ST Array (hugene10st)
Description
Primary human macrophages with a HIF-1alpha or HIF-2alpha knockdown were pretreated with IL-10 for 16h and afterwards for 4h additionaly under hypoxi (1% O2), RNA was isolated usind the Qiagen RNAeasy Kit and cDNA synthesis wos done using Ambion WT Expression Kit. Expression was compared to si control under control conditions.
Publication Title
Genome-wide identification of hypoxia-inducible factor-1 and -2 binding sites in hypoxic human macrophages alternatively activated by IL-10.
Sample Metadata Fields
Specimen part
View Samples- Mus musculus
- 8 Downloadable Samples
- Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
Analysis of newborn mouse epidermis lacking the expression of Insulin receptor (IR) and Insulin like growth factor 1 receptor (IGF-1R). Results show that IR/IGF-1R signalling control epidermal morphogenesis.
Publication Title
Insulin/IGF-1 controls epidermal morphogenesis via regulation of FoxO-mediated p63 inhibition.
Sample Metadata Fields
No sample metadata fields
View Samples- Drosophila melanogaster
- 4 Downloadable Samples
- Illumina HiSeq 2500, NextSeq 500
Description
Promoter-proximal pausing of RNA polymerase II (Pol II) is a widespread transcriptional regulatory step across metazoans. Here we find that the nuclear exon junction complex (pre-EJC) is a critical and conserved regulator of this process. Depletion of pre-EJC subunits leads to a global decrease in Pol II pausing and to premature entry into elongation. This effect occurs, at least in part, via non-canonical recruitment of pre-EJC components at promoters. Failure to recruit the pre-EJC at promoters results in increased binding of the positive transcription elongation complex (P-TEFb) and in enhanced Pol II release. Notably, restoring pausing is sufficient to rescue exon skipping and the photoreceptor differentiation defect associated with depletion of pre-EJC components in vivo. We propose that the pre-EJC serves as an early transcriptional checkpoint to prevent premature entry into elongation, ensuring proper recruitment of RNA processing components that are necessary for exon definition. Overall design: polyA mRNA -seq in conditions with the indicated knockdown treatments
Publication Title
Promoter-proximal pausing mediated by the exon junction complex regulates splicing.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples- Homo sapiens
- 12 Downloadable Samples
- Affymetrix Human Gene 1.0 ST Array (hugene10st)
Description
10 days old tumor spheroids were processed for RNA isolation using the Quiagen RNeasy Micro Kit and cDNA synthesis was done using the Ambion WT Expression Kit. Wt, HIF-1 k/d and HIF-2 k/d samples were compared to each other.
Publication Title
HIF-2alpha-dependent PAI-1 induction contributes to angiogenesis in hepatocellular carcinoma.
Sample Metadata Fields
Specimen part, Cell line
View Samples- Mus musculus
- 98 Downloadable Samples
- Illumina HiSeq 2000
Description
Acinar cells make up the majority of all cells in the pancreas, yet the source of new acinar cells during homeostasis remains unknown. Using multicolor lineage-tracing and organoid-formation assays, we identified the presence of a progenitor-like acinar cell subpopulation. These cells have long-term self-renewal capacity, albeit in a unipotent fashion. We further demonstrate that binuclear acinar cells are terminally differentiated acinar cells. Transcriptome analysis of single acinar cells revealed the existence of a minor population of cells expressing progenitor markers. Interestingly, a gain of the identified markers accompanied by a transient gain of proliferation was observed following chemically induced pancreatitis. Altogether, our study identifies a functionally and molecularly distinct acinar subpopulation and thus transforms our understanding of the acinar cell compartment as a pool of equipotent secretory cells. Overall design: The single-cell RNA-seq library preparation protocol was based on the SMART seq2 protocol (Picelli et al., 2014) with following modifications. Acinar cells were isolated as described in the section Acinar Cell Isolation and Culture and resuspended in DPBS without Ca2+ and Mg2+ (PAN-Biotech). Cells were collected in a volume of 0.5 µL and transferred to a reaction tube containing 4 µL of 6 M guanidine-HCl (Sigma-Aldrich), 0.1% (v/v) Triton X-100 (Sigma-Aldrich) and 1% (v/v) 2-mercaptoethanol (?Sigma-Aldrich). The tube was immediately transferred into liquid nitrogen and kept there for the duration of cell collection. Next, 2.2× RNA SPRI beads (Beckman Coulter) were added directly to the lysis buffer and incubated for 5 min at room temperature. The beads were washed twice with 70% ethanol. Air-dried beads were resuspended in a solution containing 2 µL of H20, 1 µL of oligo(dT) primer, and 1 µL of dNTP Mix (primer and nucleotides used as in Picelli et al., 2014). Twenty-four cells contained ERCC Spike-In RNAs (1:10,000; Mix2, Ambion) Mix in addition to primer and nucleotides. Beads were incubated for 3 min at 72°C, and reverse transcription and PCR (19 cycles) were performed as described by Picelli et al. (2014). PCR product was cleaned up using 0.8× DNA SPRI beads (Beckman Coulter), and air-dried beads were resuspended in 15 µL of H2O. The quality of cDNA library was assessed for each cell on a high-sensitivity DNA Bioanalyzer chip. Subsequent steps (tagmentation, amplification, multiplexing) were done as previously described (Llorens-Bobadilla et al., 2015). The DKFZ Genomics and Proteomics Core Facility conducted sequencing on an Illumina HiSeq2000 sequencer (paired-end 100 bp).
Publication Title
Single-Cell Analysis Uncovers Clonal Acinar Cell Heterogeneity in the Adult Pancreas.
Sample Metadata Fields
Sex, Specimen part, Cell line, Subject
View Samples- Homo sapiens
- 12 Downloadable Samples
- Affymetrix Human Gene 1.0 ST Array (hugene10st)
Description
HIF-1 is an important transcription factor for immune responses to bacterial infection. We wanted to analyze the HIF-1 dependent gene expression upon S. aureus infection and analyzed the gene expression of HepG2 nt and HepG2 HIF-1-/- cells four hours upon infection using affymetrix human gene 1.0 st. gene arrays.
Publication Title
Hypoxia-inducible factor 1-regulated lysyl oxidase is involved in Staphylococcus aureus abscess formation.
Sample Metadata Fields
Specimen part
View Samples- Mus musculus
- 4 Downloadable Samples
- Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
Global energy balance in mammals is controlled by the actions of circulating hormones that coordinate fuel production and utilization in metabolically active tissues. Bone-derived osteocalcin, in its undercarboxylated, hormonal form, regulates fat deposition and is a potent insulin secretagogue. Here, we show that insulin receptor (IR) signaling in osteoblasts controls osteoblast development and osteocalcin expression by suppressing the Runx2 inhibitor Twist-2. Mice lacking IR in osteoblasts have low circulating undercarboxylated osteocalcin and reduced bone acquisition due to decreased bone formation and deficient numbers of osteoblasts. With age, these mice develop marked peripheral adiposity and hyperglycemia accompanied by severe glucose intolerance and insulin resistance. The metabolic abnormalities in these mice are improved by infusion of exogenous under-carboxylated osteocalcin. These results indicate the existence of a bone-pancreas endocrine loop through which insulin signaling in the osteoblast ensures osteoblast differentiation and stimulates osteocalcin production, which in turn regulates insulin sensitivity and pancreatic insulin secretion to control glucose homeostasis.
Publication Title
Insulin receptor signaling in osteoblasts regulates postnatal bone acquisition and body composition.
Sample Metadata Fields
Specimen part, Time
View Samples- Mus musculus
- 3 Downloadable Samples
- Illumina HiSeq 2500
Description
2W:I-A(b) specific CD4+ T cells were isolated from immunized knock-out mice and wild-type controls on day 7 post immunization and the gene expression profiles of the cells were compared Overall design: Antigen-specifc CD4+ T cells were isolated and pooled from 4 independent experiments. The samples represent antigen-specific T cells from 15-30 mice per genotype.
Publication Title
Signaling through the adaptor molecule MyD88 in CD4+ T cells is required to overcome suppression by regulatory T cells.
Sample Metadata Fields
No sample metadata fields
View Samples