Analysis of mRNA from Gli1 expressing stromal cells of mice given SAG21k versus vehicle
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE88994 | GEO | 2016/10/21
SECONDARY ACCESSION(S): PRJNA349469
REPOSITORIES: GEO
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Project description:A single-cell transcriptional analysis was performed on GLI1+ stromal cells from the adult murine lung during homeostasis and after fibrotic injury. The goal is to understand the role of GLI1+ stromal cells in lung fibrosis and repair. Whole adult murine lung tissue from two samples were separately dissociated to single cells and subjected to fluorescence activated cell sorting (FACS) to select all live GLI1+ cells. One sample was treated with bleomycin to induce fibrosis and the other was treated with saline as a control. The single cell RNA-sequencing library was generated separately for the bleomycin and saline-treated samples. Cells were sequenced at a depth of ~70,000 reads/cell. We captured approximately 17,700 cells with a median of 2,400 genes detected per cell utilizing a droplet-based barcoding approach to capture single cells for RNA sequencing. After bleomycin-induced fibrosis, we identified a novel "myofibroblast" subset of GLI1 cells that contribute to injury and repair. Injured GLI1 cells also reveal dysregulation in key developmental pathways, including BMP signaling, that contribute to metaplastic repair after fibrotic injury.
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Project description:To assess if Hedgehog (Hh) responding cells in the skin have a unique expression profile, isolated keratinocytes that express the Hh response gene Gli1 were collected by FACS and their gene expression was compared to sorted CD34-expressing cells from the middle bulge region of the hair follicle and to cells from the interfollicular epidermis (IFE) by hybridization of isolated RNA to gene expression microarrays. Dissociated keratinocytes isolated form back skin of Gli1-eGFP/+ mice (2 mice pooled for each replicate) in adult telogen were sorted based on GFP expression and immunostaining for CD34. Only viable, single cells with immunostaining for α6 Integrin (to mark basal keratinocytes) were collected. GFP(+) cells were collected as the Gli1 cohort. GFP(-) CD34(+) cells were collected as the CD34 cohort, and GFP(-) CD34(-) cells were collected as interfollicular epidermis (IFE) cohort. Total RNA was extracted from each cell population and labeled for hybridization to gene expression microarrays. The experiment was preformed in triplicate, however RNA from the Gli1(+) cells in one replicate was of insufficient quality to analyze. For staining of isolated keratinocytes, cells were incubated on ice for 1 hour in SMEM containing 1%BSA and 15μL of anti-CD49f-PE antibody (BDBioscience) plus 5μL anti-CD34-APC antibody (eBioscience) for each 100μL total volume for each 2 million cells. Immediately prior to sorting, DAPI (Invitrogen) was added to the washed cells at 1μg/mL final concentration.
2011-12-01 | E-GEOD-28108 | biostudies-arrayexpress
Project description:To assess if Hedgehog (Hh) responding cells in the skin have a unique expression profile, isolated keratinocytes that express the Hh response gene Gli1 were collected by FACS and their gene expression was compared to sorted CD34-expressing cells from the middle bulge region of the hair follicle and to cells from the interfollicular epidermis (IFE) by hybridization of isolated RNA to gene expression microarrays.
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Project description:We sought to determine the effects of over-expression of Gli1 on gene expression in C2C12 myotube cultures. C2C12 myoblasts were induced to differentiate for 4 days. At that time, when >80% of nuclei were incorporated into multi-nucleated syncitial myotubes, we infected the cultures with recombinant adenovirus expressing GFP alone or GFP and a full length human Gli1. Media was changed 12 hours later. Cultures were lysed 60 hours after the initial infection. Gli1 over-expression induces de-differentiation of myotubes and proliferation of myoblasts. Results provide insight into the molecular basis of SHH signaling on skeletal muscle cells.
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Project description:This goal of this microarray analysis is to determine whether the mesonephros-derived theca cells exhibt a different gene expression profile from that of the whole theca cell population The mesonephros-derived (n=3, Tamoxifen at E12.5, E14.5 and E16.5) and the neonatal ovary-derived Gli1-positive cells (n=3 Tamoxifen at P1-3 via lactating dams), were isolated from the adult ovaries of Gli1-CreERT2; Rosa-LSL-tdTomato mice at 2 months of age and were sorted by FACS
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Project description:Tomato-traced single cells from the interfollicular epidermis including the touch dome (Gli1-Tomato+/Sca1+) and the hair follicle (Gli1-Tomato+/Sca1-) were FACS-sorted, randomly sequenced and clustered into epidermal (sub-)populations.
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Project description:We sought to determine the effects of over-expression of Gli1 on gene expression in C2C12 myotube cultures. C2C12 myoblasts were induced to differentiate for 4 days. At that time, when >80% of nuclei were incorporated into multi-nucleated syncitial myotubes, we infected the cultures with recombinant adenovirus expressing GFP alone or GFP and a full length human Gli1. Media was changed 12 hours later. Cultures were lysed 60 hours after the initial infection. Gli1 over-expression induces de-differentiation of myotubes and proliferation of myoblasts.
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Project description:Changes in the GLI1 cistrome following ionizing radiation were investigated using ChIP-Seq in SUM1315 triple-negative breast cancer cells.
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Project description:GLI1 is a transcription factor correlated to decreased survival in several cancers. We have identified SMARCA2 as a co-regulator that enhances GLI1-mediated transcriptional activity and functions through the C-terminal transcriptional activation domain of GLI1. Central domains including the ATPase motif of SMARCA2 physically interact with GLI1. Evaluation of DNA density indicates GLI1, like SMARCA2, can increase the DNA accessibility with a preference for sites distal to gene transcription start sites and outside the promoter regions (i.e. enhancers). The putative enhancers where accessibility is decreased by the knock down of GLI1 and SMARCA2 are located cis to genes, such as HHIP, that are regulated by GLI1 and implicated in cancer functions. At the putative enhancer for HHIP, the localization of SMARCA2 is at least partially dependent on GLI1’s presence. Understanding this transcriptional regulation by GLI1 and SMARCA2 through altering chromatin accessibility at enhances can provide additional therapeutic targets for cancers dependent on GLI1.
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