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SRP078537
Danio rerio
24 Downloadable Samples
IlluminaHiSeq2500
Description
Spermatogonial stem cells are quiescent, undergo self-renewal or differentiating divisions, thereby forming the cellular basis of spermatogenesis. This cellular development is orchestrated by follicle-stimulating hormone (FSH), through the production of Sertoli cell-derived factors, and by Leydig cell-released androgens. Here, we investigate the transcriptional events induced by Fsh in a steroid-independent manner on the restart of zebrafish (Danio rerio) spermatogenesis ex vivo, using testis from adult males where type A spermatogonia were enriched by estrogen treatment in vivo. Under these conditions, RNA sequencing preferentially detected differentially expressed genes in somatic/Sertoli cells. Fsh-stimulated spermatogonial proliferation was accompanied by modulating several signaling systems (i.e. Tgf-ß, Hedgehog, Wnt and Notch pathways). In silico protein-protein interaction analysis indicated a role for Hedgehog family members potentially integrating signals from different pathways during fish spermatogenesis. Moreover, Fsh had a marked impact on metabolic genes, such as lactate and fatty acid metabolism, or on Sertoli cell barrier components. Fish Leydig cells express the Fsh receptor and one of the most robust Fsh-responsive genes was insulin-like 3 (insl3), a Leydig cell-derived growth factor. Follow-up work showed that recombinant zebrafish Insl3 mediated pro-differentiation effects of Fsh on spermatogonia in an androgen-independent manner. Our experimental approach allowed focusing on testicular somatic genes in zebrafish and showed that the activity of signaling systems known to be relevant in stem cell systems was modulated by Fsh, providing promising leads for future work, as exemplified by the studies on Insl3. Overall design: 12 samples in total were analyzed: 6 biological replicates from control testis samples and 6 biological replicates from Fsh-treated testis samples (all co-incubated with trilostane).
Publication Title
Expression profiling identifies Sertoli and Leydig cell genes as Fsh targets in adult zebrafish testis.
Sample Metadata Fields
No sample metadata fields
View Samples- Danio rerio
- 30 Downloadable Samples
- Illumina HiSeq 2500
Description
After an acclimatization period with increasing temperature (from 27 to 35째C; ~1째C increment/day), adult zebrafish males were exposed to 35째C for 14 days and injected with the cytostatic agent busulfan (single intraperitoneal injection after 7 days at 35째C; 40 mg/Kg). Then, fish were placed back to normal water temperature and testis samples collected at different time points. Morphological analysis of testicular samples showed maximum germ cell depletion 10 days post busulfan injection (i.e. 10 dpi) and the recovery of endogenous spermatogenesis ~14 dpi. Total RNA was isolated from (1) testes of untreated adult control zebrafish, (2) germ cell-depleted, and (3) testis tissue at the beginning of the recovery period, and selected samples were used for library preparation Overall design: 15 samples in total were analyzed: 5 biological replicates from control testis samples, 5 biological replicates from depleted testis samples and 5 biological replicates from recovering testis samples
Publication Title
Endocrine and local signaling interact to regulate spermatogenesis in zebrafish: follicle-stimulating hormone, retinoic acid and androgens.
Sample Metadata Fields
Specimen part, Subject
View Samples- Mus musculus
- 23 Downloadable Samples
Affymetrix Mouse Genome 430A 2.0 Array (mouse430a2)
Description
During malaria infection is observed a robust immune response culminating on release of inflammatory mediators. This exacerbated immune response is involved in malaria symptoms and mortality. There are evidences that this response is mediated by innate immunity where pattern recognition receptors have a key role. We used microarrays to elucidate some pro-inflammatory genes that are differential expressed during P. chabaudi infection, a malarial murine model
Publication Title
Daily Rhythms of TNFα Expression and Food Intake Regulate Synchrony of Plasmodium Stages with the Host Circadian Cycle.
Sample Metadata Fields
Sex, Age, Specimen part
View Samples- Mus musculus
- 8 Downloadable Samples
- Illumina HiSeq 2500
Description
The aim of this study is to evaluate the effect of Autoimmune regulator (Aire) gene disruption in a murine medullary thymic epithelial cells (mTEC 3.10 cell line) on the transcriptome of these cells during its adhesion with thymocytes. The mTEC-thymocyte adhesion is a crucial step for the negative selection of autoreactive thymocytes and prevention of autoimmune diseases. To generate Aire mutant cell clones, a total of 5x10^5 mTEC 3.10 cells were electro-transfected (Lonza Nucleofector) with CRISPR-Cas9 plasmid targeting the Aire Exon 3 (plasmid "all in one" encoding Aire Exon 3 gRNA + Cas9 + GFP, from Sigma-Aldrich). The GFP positive mTEC single cells were sorted by using a FACS Aria III cytometer and cells were cloned by expansion in culture. Sanger sequencing of PCR products from the Aire Exon 3 of these clones was used in order to evaluate the occurrence of indel mutations within the targeted Exon 3. The mTEC 3.10 clone E6 was identified and validated as a compound heterozygous Aire KO (Aire +/-). This clone features the Aire allele 1 that encodes a mutant Aire protein carring a neutral aminoacid substitution (A118P) and allele 2 encoding a truncated Aire protein. Wild type (WT) mTEC 3.10 cells or mTEC 3.10 clone E6 were cultured in the presence (or not) of thymocytes in order to establish cell adhesion. The total RNA preparations from WT or clone E6 mTEC cells (before or after mTEC- thymocyte co-cultures) were then sequenced through RNA-sequencing using a Illumina HiSeq 2500 instrument and the TruSeq Stranded mRNA Library Preparation kit resulting in about 50 million paired-end stranded specific 100 bp reads per sample. Sequencing reads were mapped to Mus musculus reference genome (mm10) using STAR v.2.5.0a. Read counts over transcripts were calculated using HTSeq v.0.6.1p2 based on a current UCSC annotation file for GRCm38/mm10 (Dec. 2011). Overall design: The mRNA profiles of mTEC 3.10 cells carring WT Aire (before or after co-culture with thymocytes) or heterozygous KO mTEC 3.10 cells (clone E6, Aire +/-) (before or after co-culture with thymocytes) were generated by sequencing, in duplicates, using a Illumina HiSeq 2500 instrument.
Publication Title
Aire Disruption Influences the Medullary Thymic Epithelial Cell Transcriptome and Interaction With Thymocytes.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples- Homo sapiens
- 30 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
Background: Breast cancer patients present lower 1,25(OH)2D3 or 25(OH)D3 serum levels than unaffected women. Although 1,25(OH)2D3 pharmacological concentrations of 1,25(OH)2D3 may exert antiproliferative effects in breast cancer cell lines, much uncertainty remains about the effects of calcitriol supplementation in tumor specimens in vivo. We have evaluated tumor dimension (ultrassonography), proliferative index (Ki67 expression), 25(OH)D3 serum concentration and gene expression profile, before and after a short term calcitriol supplementation (dose to prevent osteoporosis) to post-menopausal patients. Results: Thirty three patients with operable disease had tumor samples evaluated. Most of them (87.5%) presented 25(OH)D3 insufficiency (<30 ng/mL). Median period of calcitriol supplementation was 30 days. Although tumor dimension did not vary, Ki67 immunoexpression decreased after supplementation. Transcriptional analysis of 15 matched pre/post-supplementation samples using U133 Plus 2.0 GeneChip (Affymetrix) revealed 18 genes over-expressed in post-supplementation tumors. As a technical validation procedure, expression of four genes was also determined by RT-qPCR and a direct correlation was observed between both methods (microarray vs PCR). To further explore the effects of near physiological concentrations of calcitriol on breast cancer samples, an ex vivo model of fresh tumor slices was utilized. Tumor samples from another 12 post-menopausal patients were sliced and treated in vitro with slightly high concentrations of calcitriol (0.5nM), that can be attained in vivo, for 24 hours In this model, expression of PBEF1, EGR1, ATF3, FOS and RGS1 was not induced after a short exposure to calcitriol. Conclusions: In our work, most post-menopausal breast cancer patients presented at least 25(OH)D3 insufficiency. In these patients, a short period of calcitriol supplementation may prevent tumor growth and reduce Ki67 expression, probably associated with discrete transcriptional changes. This observation deserves further investigation to better clarify calcitriol effects in tumor behavior under physiological conditions.
Publication Title
Calcitriol supplementation effects on Ki67 expression and transcriptional profile of breast cancer specimens from post-menopausal patients.
Sample Metadata Fields
Sex, Age, Specimen part
View Samples- Rattus norvegicus
- 6 Downloadable Samples
- Affymetrix Rat Genome 230 2.0 Array (rat2302)
Description
Polycomb-group proteins form multimeric protein complexes involved in transcriptional silencing. The Polycomb Repressive complex 2 (PRC2) contains the Suppressor of Zeste-12 protein (Suz12) and the histone methyltransferase Enhancer of Zeste protein-2 (Ezh2). This complex, catalyzing the di- and tri-methylation of histone H3 lysine 27, is essential for embryonic development and stem cell renewal. However, the role of Polycomb-group protein complexes in the control of the intestinal epithelial cell (IEC) phenotype is not known. We investigated the impact of Suz 12 depletion on gene expression in IEC-6 cells.
Publication Title
The histone H3K27 methylation mark regulates intestinal epithelial cell density-dependent proliferation and the inflammatory response.
Sample Metadata Fields
Cell line
View Samples- Mus musculus
- 18 Downloadable Samples
- Illumina HiSeq 2000
Description
Mouse lung epithelial subpopulations (alveolar type 2, basal and airway luminal cells) freshly dissociated from mouse lung and trachea were isolated by FACS. RNA-seq gene expression profiling was used to determine gene signature from each population. Overall design: Cells were isolated from the small airway (SA) and large airway (LA) of 6 mouse lungs
Publication Title
Lung Basal Stem Cells Rapidly Repair DNA Damage Using the Error-Prone Nonhomologous End-Joining Pathway.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples- Mus musculus
- 13 Downloadable Samples
- Illumina HiSeq 2500
Description
The specification of hematopoietic cells in the developing embryo occurs in specific stages and is regulated by the successive establishment of specific transcriptional networks. However, the molecular mechanisms of how the different stages switch from one to another are still not well understood. Hematopoietic cells arise from endothelial cells within the dorsal aorta which transit into hematopoietic cells by a process called the endothelial-hematopoietic transition (EHT) which does not involve DNA replication. The transcription factor RUNX1 is essential for this process. Using the differentiation of mouse embryonic stem cells carrying an inducible version of RUNX1, we have previously shown that hematopoietic genes are primed prior to the EHT by the binding of transcription factors required to form both endothelial and hematopoietic cells (FLI-1 and SCL/TAL1). We demonstrated that after induction RUNX1 reshapes the transcription factor binding landscape by causing a relocation of these factors and pulling them towards its binding sites. In the study presented here, we employed the same system to globally dissect the transcriptional processes that underlay the EHT. We demonstrate that the RUNX1-mediated movement of FLI-1 involves the recruitment of the basal transcription components CDK9 and BRD4 to promoters. The looping factor LDB1 to binds to distal elements and after induction relocates towards RUNX1/FLI-1 to form a co-localizing complex in chromatin. This entire process is blocked by treatment with the BRD4 inhibitor JQ1. Our study constitutes a paradigm for transcriptional processes driving transitions in cellular shape and function which are widely observed in development and disease. Overall design: RNA-seq expreiments have been used to study RUNX1 transcription factor during Hematopoietic specification
Publication Title
The Co-operation of RUNX1 with LDB1, CDK9 and BRD4 Drives Transcription Factor Complex Relocation During Haematopoietic Specification.
Sample Metadata Fields
Specimen part, Subject
View Samples- Mus musculus
- 6 Downloadable Samples
- Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
Histone deacetylases (Hdac) remove acetyl groups from proteins, influencing global and specific gene expression. Hdacs control inflammation, as shown by Hdac inhibitor-dependent protection from DSS-induced murine colitis. While tissue-specific Hdac knockouts show redundant and specific functions, little is known of their intestinal epithelial cell (IEC) role. We have shown previously that dual Hdac1/Hdac2 IEC-specific loss disrupts cell proliferation and determination, with decreased secretory cell numbers and altered barrier function. We thus investigated how compound Hdac1/Hdac2 or Hdac2 IEC-specific deficiency alters the inflammatory response. Floxed Hdac1 and Hdac2 and villin-Cre mice were interbred. Compound Hdac1/Hdac2 IEC-deficient mice showed chronic basal inflammation, with increased basal Disease Activity Index (DAI) and deregulated Reg gene colonic expression. DSS-treated dual Hdac1/Hdac2 IEC-deficient mice displayed increased DAI, histological score, intestinal permeability and inflammatory gene expression. In contrast to double knockouts, Hdac2 IEC-specific loss did not affect IEC determination and growth, nor result in chronic inflammation. However, Hdac2 disruption protected against DSS colitis, as shown by decreased DAI, intestinal permeability and caspase-3 cleavage. Hdac2 IEC-specific deficient mice displayed increased expression of IEC gene subsets, such as colonic antimicrobial Reg3b and Reg3g mRNAs, and decreased expression of immune cell function-related genes. Our data show that Hdac1 and Hdac2 are essential IEC homeostasis regulators. IEC-specific Hdac1 and Hdac2 may act as epigenetic sensors and transmitters of environmental cues and regulate IEC-mediated mucosal homeostatic and inflammatory responses. Different levels of IEC Hdac activity may lead to positive or negative outcomes on intestinal homeostasis during inflammation
Publication Title
The acetylome regulators Hdac1 and Hdac2 differently modulate intestinal epithelial cell dependent homeostatic responses in experimental colitis.
Sample Metadata Fields
Specimen part
View Samples- Mus musculus
- 6 Downloadable Samples
- Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
Acetylation and deacetylation of histones and other proteins depend on the opposing activities of histone acetyltransferases and histone deacetylases (HDACs), leading to either positive or negative gene expression changes. The use of HDAC inhibitors (HDACi) has uncovered a role for HDACs in the control of proliferation, apoptosis and inflammation. However, little is known of the roles of specific HDACs in intestinal epithelial cells (IEC). We investigated the consequences of ablating both Hdac1 and Hdac2 in murine IECs gene expression.
Publication Title
HDAC1 and HDAC2 restrain the intestinal inflammatory response by regulating intestinal epithelial cell differentiation.
Sample Metadata Fields
Specimen part
View Samples