Expression data from a mouse small cell lung cancer (SCLC) cell line, KP1, transfected with Notch-ICD
ABSTRACT: Transient transfection of activated Notch1 (Notch1-ICD) decreases cellular proliferation and reduces the expression of a subset of neuroendocrine genes. We used microarrays to identify the gene expression changes 48 hours after overexpression of Notch1-ICD. mSCLC cells transfected with Notch1-ICD-ires-GFP were FACS sorted for GFP+ cells 48 hours after transfection. RNA was then extracted and hybridized to Affymetrix GeneChip® Mouse Gene 2.0 ST array.
Project description:Convergent and divergent effects of two ICD ERBB4 isoforms on gene expression in normal-like MCF10A mammary epithelial cells lacking endogeneous ERBB4 expression. Control vector (VEC) and ERBB4 expression MCF10A cells (ICD CYT1 or CYT2) maintained in regular medium containing 10% serum, were collected and RNA samples were collected and analyzed.
Project description:We evalueted gene expression in dorsal root ganglion (DRG) over-expressing NRG1 typeIII ICD (Intra Cellular Domain). DRG were infected with a lentivirus expressing ICD and compared to not infected DRG or DRG infected with a lentivirus expressing EGFP.
Project description:The Notch signaling pathway functions in a number of processes during embryologic development, especially the maintenance or aquisition of cell fate. We purturb the Notch signalling pathway in embryonic Xenopus laevis in order to 1) better characterize the downstream targets of Notch signalling, and 2) determine the extent to which early embryos can recover from transient purturbations to critical signalling pathways, if at all. Xenopus laevis embryos were unilaterally injected at the two cell stage with either GFP, GFP and ICD (Notch intracellular domain, an up-regulator of the Notch pathway), or GFP and DBM (domain-binding mutant, a downregulator of the Notch pathway). At stages 18, 28, and 38, for each injection, pooled total RNA from 10 embryos was extracted. Extraction was performed for three biological replicates for each time/injection condition. cDNA from total RNA was hybridized on Affymetrix Xenopus laevis Genome 2.0 arrays.
Project description:We identified histidine triad nucleotide binding protein 1 (HINT1) as a human teneurin-1 ICD interaction partner in a yeast-2 hybrid screen. This interaction was confirmed in human cells, where HINT1 is known to inhibit the transcription of target genes by directly binding to transcription factors at the promoter. In a whole transcriptome analysis of BS149 glioblastoma cells overexpressing the teneurin-1 ICD, several microphthalmia-associated transcription factor (MITF) target genes were found to be up-regulated. Interestingly, MITF is one of the transcription factors inhibited by HINT1. Thus, we directly compare the transcriptomes of MITF versus TEN1-ICD overexpressing BS149 cells in this study, in order to reveal any co-regulated genes. For the whole transcriptome analysis of MITF, cells were transiently transfected in triplicates with either, pcDNA3.1-RFP-HA (negative control) or pcDNA3.1-MITF-RFP-HA. The overexpressing cells were then FACS-sorted directly into RLT lysis buffer (Qiagen) at a 3:1 volume ratio of lysis buffer to cells in PBS, 24 h post-transfection.
Project description:Teneurins are large type II transmembrane proteins that are necessary for the normal development of the central nervous system (CNS). While many studies highlight the significance of teneurins, especially during development, there is only limited information known about the molecular mechanisms of function. Previous studies have shown that the N-terminal intracellular domain (ICD) of teneurins can be cleaved at the membrane and subsequently translocates to the nucleus where it can influence gene transcription. Target genes as well as mechanisms have yet to be elucidated, and thus we are investigating the transcriptional activity of the human teneurin-1 ICD in this study. For the whole transcriptome analysis of TEN1-ICD overexpression, we used a modified and improved tet-system. Two separate vectors are required to make the cell line stable. One contains the tet-activating domain fused to a glucocorticoid binding domain (GBD). The other contains the tetO operator sequences directly upstream of a CMV promoter and the gene to be overexpressed. BS149 cells were first transfected with pirtetR-GBD and made stable by Puromycin selection, and then after further transfection with either ptetO-eGFP-His (negative control) or ptetO-TEN1-ICD-eGFP-His by Hygromycin selection. The stable BS149 cell lines were split into three 10 cm Petri dishes each. The triplicate cell lines were cultured once before induction with Dexamethasone and Doxycycline. The overexpressing cells were then FACS-sorted directly into RLT lysis buffer (Qiagen) at a 3:1 volume ratio of lysis buffer to cells in PBS, 24 h post-induction.
Project description:Ikaros family DNA binding proteins are critical regulators of B cell development. To identify Ikaros-regulated genes in pre-B cells we performed gene expression studies at enhanced temporal resolution. We used retroviral gene transfer to express Ikaros proteins in the pre-B cell line B3. Total RNA from 2 biological replicates of B3 cells transduced with wild type Ikaros (HA-Ikaros-IRES-GFP) and DNA binding-deficient Ikaros mutant 159A (HA-159A Ikaros-IRES-GFP) was isolated 48h after infection. To increase the temporal resolution of Ikaros-regulated gene expression we transduced B3 cells with inducible Ikaros (HA-Ikaros-ERt2-IRES-GFP) and isolated total RNA from 3 biological replicates after 2 h and 6 h of exposure to 4-hydroxytamoxifen. Vector transduced B3 cells (ERt2-IRES-GFP) treated with 4-hydroxytamoxifen were used as controls.
Project description:The most recurrently mutated oncogene in T-cell acute lymphoblastic leukemia (T-ALL) is NOTCH1. The core Notch complex consists of an ICN protein, a Maml cofactor, and the DNA binding factor Rbpj. The known direct cofactors of Notch appear to act nonselectively, homogeneously driving Notch gene expression functions. It is unclear whether there are direct cofactors of Notch that act selectively and heterogeneously regulate ICN. We discovered that Zmiz1, a Protein Inhibitor of Activated STAT (PIAS)-like coactivator, directly bound ICN1. ChIP-Seq showed that Zmiz1 selectively co-bound only a subset of Notch-regulated enhancers. This led to hypothesize that Zmiz1 regulates only a subset of Notch1 target genes. To investigate this, we performed RNA-Seq on four 8946 cell linesin which L1601P (activated Notch1) or Zmiz1 were expressed alone or in combination. Zmiz1 induced ~10% of Notch target genes. The Notch target gene that was most strongly induced by Zmiz1 was Myc. Our data suggest that Zmiz1 selectively amplifies a subset of Notch target genes with strong amplification of Myc. RNA-Seq in a murine T-ALL cell line
Project description:Ikaros family DNA binding proteins are critical regulators of B cell development. To identify Ikaros-regulated genes in primary pre-B cells we performed gene expression microarrays. We used retroviral gene transfer to express Ikaros proteins. Total RNA from 3 biological replicates of primary pre-B cells transduced with wild type Ikaros (HA-Ikaros-IRES-GFP) and control vector (IRES-GFP) was isolated 48h after infection.
Project description:Ires-GFP and CITED4-ires-GFP overexpressing permanent cell lines were generated by transfection of the CRC cell line SW480 with either the ires-GFP containing vector vector (in pCDNA4HISMAX) or the CITED4-ires GFP in (pCDNA4HISMAX) and selected with 2 ug/ ml puromycin in order to prepare permanent cell lines. For microarray analysis, the two cell lines were plated at a density of 1 x 10E5 cell in 12 well plates in 1 ml RPMI plus 10% FCS and penicillin/ streptomycin. The respective cell lines were harvested at day 1, 3 and 5 for RNA preparation and microarray analysis using Agilent human 4 x 44k expression microarrays. Every time point (cy3 labeled) was hybridzed together with Stratagene Human Total RNA standard as a control (cy5). Comparisons were made between the CITED4 overexpressing permanent cell line and ires-GFP containing cell line.
Project description:The goal was to study the role of Hlx in hematopoiesis. Sorted Lin-Kit+Sca-1+ cells from wild-type FVB/nJ bone marrow were infected with control (pCAD-IRES-GFP) or Hlx lentivirus (pCAD-IRES-GFP-Hlx) and cultured for 2 days in Iscove’s modified Dulbecco’s medium (IMDM) containing FBS, mIL-3, mIL-6 and mSCF with lentiviral supernatants in the presence of 8ug/ml polybrene. Subsequently, GFP+ cells were sorted by FACS and RNA was prepared. Three replicates of each, control vector transduced and HLX-transduced cells were used.