Expression profile of oesophageal epithelium basal cells comparing DNMaml expressing cells versus controls
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Jo Fowler
PROVIDER: E-MTAB-2334 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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2012-02-22 | E-GEOD-34961 | biostudies-arrayexpress
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Project description:MLL-fusions may induce leukemogenic gene expression programs by recruiting the histone H3K79 methyltransferase to MLL-target promoters. We evaluated gene expression changes after cre-mediated loss of Dot1l in leukemia cells obtained from mice injected with MLL-9 transformed lineage negative bone marrow cells. MLL-AF9 murine leukemia cells carrying two conditional Dot1l alleles were retrovirally transduced with Cre or empty control vector, and gene expression changes were monitored on day 3, 5, and 7 after transduction.
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Project description:Rho-of-plant small GTPases (ROPs) are regulators of cell polar growth processes and of plant-pathogen interactions. The barley ROP protein RACB is involved in susceptibility towards infection by the barley powdery mildew fungus Blumeria hordei, but little is known about the cellular pathways that connect RACB-signaling to disease susceptibility. We now used an untargeted co immunoprecipitation-liquid chromatography-coupled mass spectrometry screening from Bh-infected epidermal layers of barley plants, which expressed green fluorescing protein-tagged constitutively activated (CA) RACB. This uncovered novel RACB interaction partners of plant and fungal origin. Three of those proteins, a plant phosphoinositide phosphatase, a plant phosphoinositide phospholipase and one putative Bh-effector protein appear to be involved in the barley-Bh-pathosystem and function in disease resistance or susceptibility, respectively. RACB and its plant interactors bind to overlapping anionic phospholipid species in vitro, and in case of RACB, this lipid-interaction is mediated by its carboxy-terminal polybasic region (PBR). Four diverse fluorescent plant anionic phospholipid-markers show altered subcellular distribution during Bh-attack. Phosphatidyl-4-phosphate and phosphatidylserine show a distinct enrichment at the haustorial neck region, suggesting a connection to subcellular targeting of RACB at this site. We suggest that the interplay of ROPs with anionic phospholipids and phospholipid–metabolizing enzymes may steer subcellular enrichment of components that are pivotal for fungal penetration success or failure.
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Project description:Abelson virus (v-Abl)-transformed pre-B cell lines from BM of dcrfl/fl; R26R-yfp (4470; 4475) and dcrfl/+; R26R-yfp mice (4483) are treated with a transducible Cre protein (Tat-Cre) to induce deletion of the dicer-1 gene in vitro. Cre activity is conveniently monitored by concomitant expression of YFP. To obtain Dicer KO cells, YFP+ cells from dcrfl/fl; R26R-yfp (4470_YFP; 4475_YFP) cultures treated with Tat-cre were isolated by fluorescence-activated cell sorting (FACS) and further propagated in cell culture. As controls, we used non-deleted, YFP- cells sorted from dcrfl/fl; R26R-yfp cultures (4470; 4475) treated with Tat-cre, and YFP+ deleted cells from dcrfl/+; R26R-yfp cultures (4483_YFP). To validate this system, various cell populations were sorted and analyzed for Dicer protein and miRNA expression. In the Dicer KO pro-B cell lines, Western analysis indicated that Dicer protein was efficiently ablated, and Northern blots demonstrated that the levels of mature miR-17 and miR-191 were drastically reduced.
2008-03-08 | E-MEXP-1455 | biostudies-arrayexpress
Project description:To unravel the potential cooperative roles of oxygen-regulated signaling pathways; von Hippel-Lindau (VHL) tumor suppressor protein and hypoxia-inducible factor (HIF) transcription factors, we have generated mutant mice with; Vhlh, Vhlh/Hif1α, Vhlh/Hif2α and Vhlh/Hif1α/Hif2α gene alleles floxed. Subsequently primary kidney cells were isolated, cultured and infected with Adenoviruses bearing either Cre/GFP or GFP expression only. Agilent cDNA microarrays were utilized to compare the gene expression profiles of the kidney epithelial cells from aforementioned cell cultures to gain insight about the molecules and signaling pathways that drive aberrant cellular proliferation in clear cell renal cell carcinoma (ccRCC). 24 independent biological samples from 4 genetic experimental conditions were hybridized in two- color format on 13 Mouse GE 4x44K v2 Microarray Kit (Design ID 026655) arrays. Eventual dye specific hybridization effects were controlled with dye swap on biological replicates. For an each experimental condition 3 arrays were use, except of one where 4 arrays were used (one sample combination in repetition with a dye swap). For the final analysis the gene expression levels were dye effect corrected and values for biological replicates were averaged to obtain a matrix with gene expression levels for 4 independent biological (1. Vhlh-/-, 2. Vhlh-/-/Hif1α-/-, 3. Vhlh-/-/Hif2α-/- and 4. Vhlh-/-/Hif1α-/-/Hif2α-/-) conditions.
2016-01-08 | E-GEOD-75745 | biostudies-arrayexpress
Project description:The skin interfollicular epidermis (IFE) is the first barrier against the external environment and its maintenance is critical for survival. Two seemingly opposite theories have been proposed to explain IFE homeostasis. One posits that IFE is maintained by a long-lived slow-cycling stem cell (SC) population that give rise to short-lived transit-amplifying (TA) cell progeny, while the other suggests that homeostasis is achieved by a single committed progenitor (CP) that balances stochastic fate. Here, we probed the cellular heterogeneity within the IFE using two different inducible CREER targeting IFE progenitors. Quantitative analysis of clonal fate data and proliferation dynamics demonstrate the existence of two distinct proliferative cell compartments composed of slow-cycling SC and CP, both of which undergo population asymmetric self-renewal. However, following wounding, only SCs contribute substantially to the repair and long-term regeneration of the tissue, while CP cells make a minimal and transient contribution. Transcriptional profile of InvCREER/RosaYFP and K14CREER/RosaYFP targeted FACS-isolated alpha6 integrin-high CD34-neg basal cells from mouse tail epidermis. Skin epidermis was removed from tail bone and incubated overnight in HBSS (Gibco) 0.25% trypsin (Gibco) at 4M-BM-0C. Epidermis was separated from the dermis and incubated on a rocking plate (100 rpm) at room temperature for 5 min. Basal cells were mechanically separated from the epidermis by flushing 10 times under the epidermis. Tissues were then cut in pieces of 1 mm2 with scalpel, and trypsin was neutralized by adding DMEM medium (Gibco) supplemented with 2% Chelex Fetal Calf Serum (FCS). Samples were filtrated on 70 and 40M-BM-5m filter (Falcon). Immunostaining was performed using biotin-conjugated anti-CD34 (clone RAM34; BD Biosciences) PE-conjugated anti-M-NM-16-integrin (clone GoH3; BD biosciences). Primary antibodies were washed with 2% FCS/PBS and cells were incubated for 30 min in APC conjugated streptavidin (BD Biosciences) secondary antibodies, on ice, with shaking every 10 min. Living K14- and involucrin-expressing epidermal cells were gated by forward scatter, side scatter, negative staining for Hoechst dye and by following the YFP signal. Basal cells from the interfollicular epidermis were targeted using CD34 negative alpha 6 high gating. Fluorescence-activated cell sorting analysis was performed using FACSAria I at high pressure (70 psi) and FACSDiva software (BD Biosciences). Sorted cells were harvested directly in the lysis buffer provided by the RNeasy microkit (QIAGEN) supplemented with 1M-BM-5l of beta-mercaptoethanol for every 100M-BM-5l of lysis buffer. RNA extraction was performed on freshly sorted cells according to the manufacturerM-bM-^@M-^Ys protocol.
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