Background: The terminal duct lobular unit (TDLU) is the most dynamic structure in the human breast and the putative site of origin of human breast cancer. Although stromal cells contribute to a specialized microenvironment in many organs, this component remains largely understudied in the human breast. We here demonstrate the impact on epithelium of two lineages of breast stromal fibroblasts, one of which accumulates in the TDLU while the other resides outside the TDLU in the interlobular stroma. Methods: The two lineages are prospectively isolated by FACS based on different expression levels of CD105 and CD26. The characteristics of the two fibroblast lineages are assessed by immunocytochemical staining and gene expression analysis. The differentiation capacity of the two fibroblast populations is determined by exposure to specific differentiating conditions followed by analysis of adipogenic and osteogenic differentiation. To test whether the two fibroblast lineages are functionally imprinted by their site of origin, single cell sorted CD271low/MUC1high normal breast luminal epithelial cells are plated on fibroblast feeders for the observation of morphological development. Epithelial structure formation and polarization is shown by immunofluorescence and digitalized quantification of immunoperoxidase stained cultures. Results: Lobular fibroblasts are CD105high/CD26low while interlobular fibroblasts are CD105low/CD26high. Once isolated the two lineages remain phenotypically stable and functionally distinct in culture. Lobular fibroblasts have properties in common with bone marrow derived mesenchymal stem cells and they specifically convey growth and branching morphogenesis of epithelial progenitors. Conclusions: Two distinct functionally specialized fibroblast lineages exist in the normal human breast, of which the lobular fibroblasts have properties in common with mesenchymal stem cells and support epithelial growth and morphogenesis. We propose that lobular fibroblasts constitute a specialized microenvironment for human breast luminal epithelial progenitors, i.e. the putative precursors of breast cancer.
Evidence of two distinct functionally specialized fibroblast lineages in breast stroma.
Specimen part
View SamplesYeast lacking the H3 or H4 amino termini, and corresponding wild type strains, were grown in synthetic media. These conditions induce Gcn4-activated transcription.
Contribution of the histone H3 and H4 amino termini to Gcn4p- and Gcn5p-mediated transcription in yeast.
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View SamplesAbf1 and Rap1 are General Regulatory Factors that contribute to transcriptional activation of a large number of genes, as well as to replication, silencing, and telomere structure in yeast. In spite of their widespread roles in transcription, the scope of their functional targets genome-wide has not been previously determined. We have used microarrays to examine the contribution of these essential GRFs to transcription genome-wide, by using ts mutants that dissociate from their binding sites at 37 C. We combined this data with published ChIP-chip studies and motif analysis to identify probable direct targets for Abf1 and Rap1. We also identified a substantial number of genes likely to bind Rap1 or Abf1, but not affected by loss of GRF binding. Interestingly, the results strongly suggest that Rap1 can contribute to gene activation from farther upstream than can Abf1. Also, consistent with previous work, more genes that bind Abf1 are unaffected by loss of binding than those that bind Rap1. Finally, we showed for several such genes that the Abf1 C-terminal region, which contains the putative activation domain, is not needed to confer this peculiar "memory effect" that allows continued transcription after loss of Abf1 binding.
Genome-wide analysis of transcriptional dependence and probable target sites for Abf1 and Rap1 in Saccharomyces cerevisiae.
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View SamplesSignal intensity data for rpd3 delete, H3delta(1-28), H3(K4,9,14,18,23,27Q), H4delta(2-26), H4(K5,8,12,16Q), rpd3 delete H3delta(1-28), and rpd3 delete H4(K5,8,12,16Q) yeast grown in rich (YPD) media
Genome-wide analysis of the relationship between transcriptional regulation by Rpd3p and the histone H3 and H4 amino termini in budding yeast.
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View SamplesDifferential gene expression between groups of homogenous cell types is a biological question whose time has come. RNA can be extracted from small numbers of cells, such as those isolated by laser capture microdissection, but the small amounts obtained often require amplification to enable whole genome transcriptome profiling by technologies such as microarray analysis and RNA-seq. Recently, advances in amplification procedures make amplification directly from whole cell lysates possible. The aim of this study was to compare two amplification systems for variations in observed RNA abundance attributable to the amplification procedure for use with small quantities of cells isolated by laser capture microdissection. Arabidopsis root cells undergoing giant cell formation due to nematode infestation and un-infested control root cells were laser captured and used to evaluate 2 amplification systems. One, NuGEN's WT-Ovation Pico amplification system, uses total RNA as starting material while the other, NuGEN's WT-One-Direct Amplification system, uses lysate containing the captured cells. The reproducibility of whole genome transcript profiling and correlations of both systems were investigated after microarray analysis. The NuGEN WT-Ovation One-Direct system was less reproducible and more variable than the NuGEN WT-Ovation Pico system. The NuGEN WT-Ovation Pico Amplification kit resulted in the detection of thousands of genes differentially expressed genes between giant cells and control cells. This is in marked contrast to the relatively few genes detected after amplification with the NuGEN WT-Ovation One-Direct Amplification kit.
Comparison between NuGEN's WT-Ovation Pico and one-direct amplification systems.
Specimen part, Time
View SamplesConditional IRF8 KO mice (mice with a conditional allele of Irf8 crossed with CD19-Cre mice) showed increased numbers of both Gene expression data spleen marginal zone (MZ) and Gene expression data spleen follicular (FO) B cells compared to control mice.
IFN regulatory factor 8 restricts the size of the marginal zone and follicular B cell pools.
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View SamplesGene expression was compared for wild type yeast (BY4741) and yeast lacking Gal11/Med15 and Med3, or from a gal11-myc med3 strain. The gal11-myc allele shows a partial loss of function when combined with med3. Expression was analyzed for yeast grown in YPD as well as in CSM.
Distinct role of Mediator tail module in regulation of SAGA-dependent, TATA-containing genes in yeast.
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View SamplesPURPOSE To identify retinal genes and their relevant expression pathways affected by intravitreal injections of dexamethasone and triamcinolone acetonide in mice at clinically relevant time points for patient care.
In vivo gene expression profiling of retina postintravitreal injections of dexamethasone and triamcinolone at clinically relevant time points for patient care.
Sex, Specimen part
View SamplesPURPOSE To identify retinal pigment epithelial (RPE)/choroid genes and their relevant expression pathways affected by intravitreal injections of dexamethasone and triamcinolone acetonide in mice at clinically relevant time points for patient care. METHODS Differential gene expression of over 34,000 well-characterized mouse genes, in the RPE/choroid of 6 week old C57BL/6J mice were analyzed after intravitreal steroid injections at 1 week and 1 month post injection, using Affymetrix Mouse Genome 430 2.0 microarrays. The data were analyzed using GeneSpringGX12.5 and Ingenuity Pathway Analysis (IPA) microarray analysis software for biologically relevant changes. RESULTS Both triamcinolone and dexamethasone caused differential activation of genes involved in Circadian Rhythm Signaling pathway at both time points tested. Triamcinolone (TAA) uniquely induced significant changes in gene expression in Calcium Signaling (1 week) and Glutamate Signaling pathways (1month). In contrast, Dexamethasone (Dex) affected the GABA Receptor Signaling (1 week) and Serotonin Receptor Signaling (1month) pathways. CONCLUSIONS Understanding how intraocular steroids affect the gene expression of RPE/choroid is clinically relevant. This in vivo study has elucidated several genes and pathways that are potentially altering the circadian rhythms and several other neurotransmitter pathways in RPE/choroid cells during intravitreal steroid injections, which likely has consequences in the dysregulation of RPE function and neurodegeneration of the retina.
Comparison of In Vivo Gene Expression Profiling of RPE/Choroid following Intravitreal Injection of Dexamethasone and Triamcinolone Acetonide.
Sex, Specimen part, Treatment
View SamplesWe performed Ago HITS-CLIP to identify targets of viral and human miRNAs in latently KSHV-infected PEL cells Overall design: Ago HITS-CLIP was performed in two latently infected PEL cell lines, BCBL-1 and BC-3; Argonaute-immunoprecipitation of UV cross-linked Ago-miRNA-mRNA complexes, followed by RNA isolation, library construction, and high-throughput sequencing (Illumina GAxII); we performed 3 biological replicates for each cell line, two technical (sequencing) replicates of BCBL-1 biological replicate 1
Ago HITS-CLIP expands understanding of Kaposi's sarcoma-associated herpesvirus miRNA function in primary effusion lymphomas.
Cell line, Subject
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