Expression data from human mesenchymal stem cells treated with Wnt3a
ABSTRACT: Wnt signaling is upregulated frequently in several cancers, including sarcomas. Since, there is cell-context dependent variation in the target gene expression, to identify canonical Wnt targets in sarcomas, we used human mesenchymal stem cells. Human mesenchymal stem cells were treated with 100ng/ml recombinant Wnt3a either 6 hrs or 24 hrs. Total RNA was extracted from untreated and Wnt3a treated samples using Qiagen's RNA extraction kit.
Project description:Wnt signaling is upregulated frequently in several cancers, including sarcomas. Since, there is cell-context dependent variation in the target gene expression, to identify canonical Wnt targets in sarcomas, we used human mesenchymal stem cells. Overall design: Human mesenchymal stem cells were treated with 100ng/ml recombinant Wnt3a either 6 hrs or 24 hrs. Total RNA was extracted from untreated and Wnt3a treated samples using Qiagen's RNA extraction kit.
Project description:We analyzed the transcriptome of two different triple negative breast cancer (TNBC) cell lines to define a comprehensive list of Wnt target genes. Cells were treated with Wnt3a for 6h, 12h or 24h. We found up-regulated and down-regulated genes in response to Wnt3a treatment. They are involved in the Wnt pathway itself, and also in TGFß, p53 and Hedgehog pathways. Thorough characterization of these novel potential Wnt target genes may reveal new regulators of the canonical Wnt pathway. The comparison of our list of Wnt target genes with those published in other cellular contexts confirms the notion that Wnt target genes are tissue-, cell line- and treatment-specific. Cells were seeded in six-well plates, serum starved overnight then treated with Wnt3a for the indicated times (6, 12 and 24 hours). Triplicates for each condition were included in the experiment.
Project description:The Wnt3a/β-catenin and Activin/Smad2,3 signaling pathways synergize to induce endodermal differentiation of human embryonic stem cells, however the mechanism is not well-understood. Using ChIP-seq and GRO-seq analyses, we report here that hESC enhancers, including Wnt3a/LEF-1 sites, hold enhancer RNAPII complexes (eRNAPII) containing high levels of Ser5P and low Ser7P. In Wnt3a signaling, β-catenin recruits cohesin to the LEF-1:eRNAPII sites to induce enhancer-promoter looping and activate transcription of mesoendodermal (ME) genes. However, paused Ser5P-RNAPII complexes accumulate at these genes, indicating that elongation remains limiting. Subsequent Activin/Smad2,3 signaling increases P-TEFb occupancy, CTD-Ser7P, and productive elongation at ME genes. Additionally, ME genes, including EOMES and MIXL1, are repressed by the Hippo regulator, Yap1, an essential pluripotency factor. GRO-seq experiments indicate that Yap1 blocks nascent transcription and controls NELF occupancy on ME genes. Thus, Wnt3a/β-catenin and Activin/Smad2,3 pathways up-regulate transcription initiation and elongation, respectively, to overcome Yap1 repression during early hESC differentiation ChIP-seq and GROseq experiments in H1 hESCs. Cells were treated with Wnt3a (200ng/ml), Activin A (100ng/ml) or Wnt3a+Activin A (W200ng/ml+A100ng/ml) for 4h (ChIP-seq) or 6h (GRO-seq). GRO-seq in YAP depleted cells were carried out following transfection with control or YAP siRNAs . After 48h transfection, cells were left untreated or treated with Wnt3a+Activin (W200ng/ml+A100ng/ml) for additional 6h.
Project description:In order to gain insight into the molecular events operating downstream of canonical wnt-signaling in myoblasts, we compared by microarray analysis the transcriptome of myoblast cultured for 4 hours in the presence and absence of Wnt3a. Overall design: Primary myoblasts isolated from newborn mice were treated for 4 hours in DMEM 2% horse serum in presence or in absence of Wnt3a (100ng/ml)
Project description:Adult stem cells have the ability to self-renew and to generate specialized cells. Self-renewal is dependent on extrinsic niche factors but few of those signals have been identified. We show that adult mammary glands contain a Wnt-responsive cell population that is enriched for stem cells. In cell culture experiments, exposure to purified Wnt protein clonally expands mammary stem cells for many generations and maintains their ability to generate functional glands in transplantation assays. We propose here that Wnt3A treated mammary stem cells retain their stemness through the regulation of its downstream target genes. We used microarrays to detail the global gene expression pattern underlying mammary stem cells response towards Wnt3A treatment and identified distinct classes of genes during this process Mammary glands from 8- to 12-week-old virgin female mice were isolated and single-cell suspension was obtained. Mammary stem cell enriched population (Lin-, CD24+, CD29hi) cells were isolated using BD FACSAria. The purity of sorted population was routinely checked and ensured to be more than 95%.Total RNA from 2nd colonies passage cultured in the presence of vehicle and Wnt3A was extracted with PicoPure (Arcturs) in accordance with the manufacturer’s protocol. RNA concentration was determined with NanoDrop ND-1000, and quality was determined using the RNA 6000 Nano assay on the Agilent 2100 Bioanalyzer (Agilent Technologies). Affymetrix microarray analysis, fragmentation of RNA, labelling, hybridization to Mouse Genome 430 2.0 microarrays, and scanning were performed in accordance with the manufacturer’s protocol (Affymetrix). Total 4 samples representing two sets of replicates were analyzed.
Project description:R-spondins (Rspos) comprise a family of four secreted proteins that have important roles in cell proliferation, cell fate determination and organogenesis. Rspos typically exert their effects by potentiating the Wnt/β-catenin signaling pathway. To systematically investigate the impact of Rspo/Wnt on gene expression, we performed a microarray analysis using C57MG mouse mammary epithelial cells treated with recombinant Rspo2 and/or Wnt3a. This study compares gene expression in C57MG cells after 24h treatment with recombinant Rspo2 and recombinant Wnt3a alone or in combination versus BSA treatment. Control cells were treated with BSA, since it was used as carrier for the recombinant proteins. Each treatment was done in biological triplicate.
Project description:To test the potential of canonical Wnt signalling to modulate nociception via transcriptional regulation in dorsal root ganglion (DRG) neurons, we performed a genome-wide transcriptome analysis of neuron-enriched DRG cultures treated with Wnt3a or vehicle for 6 hours. Total RNA obtained from neuron-enriched mouse DRG culture subjected to Wnt3a treatment for 6 hours was compared to a matched control (vehicle-treated) DRG culture.
Project description:Wnt pathway is dysregulated in CLL-We characterized Wnt pathway gene expression in normal B and CLL-B cells and identified Wnt targets in normal B and CLL-B cells through this data set. In this dataset, we included normal B cells and CLL-B cells for Wnt pathway gene expression. This leads to the identification of 62 Wnt pathway components which are differnetially expressed between normal and CLl-B cells. We also included normal B cells and CLL-B cells with or without Wnt3a treatment and identified 468 and 676 Wnt regulated genes in normal and CLL B cells, respectively. 24 normal B cells and 179 CLL-B cells were used in the Wnt pathway gene expression analysis. Three normal and three CLL samples were included in the identification of Wnt regulated targets
Project description:MIXL1-GFP reporter lines were differentiated as Spin EBs in APEL medium supplemented with Wnt3a alone, BMP4 alone or Wnt3a/BMP4 in combination. EBs induced in the absence of growth factors were used as control. EBs induced with BMP4 or Wnt3a/BMP4 were FACS sorted based on E-CADHERIN and GFP expression. Unsorted EBs and sorted fractions were subjected to Illumina microarray processing.
Project description:The early transcriptional response of HeLa cervical carcinoma cells to the canonical Wnt signalling pathway was investigated at two different cell cycle phases (G1 and G2/M) via microarray gene expression profiling. HeLa cells grown under standard conditions were treated for 3 h with either Control, Wnt3a- or Dkk1-conditioned media and then FACS-sorted into G1 and G2/M populations for microarray expression analysis with 4 independent replicates per group.