Gene expression profile upon forced Notch1 activation in LS174T cells
ABSTRACT: The cell line was modified to express activated form of Notch1 (NICD1 of mouse origin) upon Doxycycline addition to the culture medium. Generation was performed by using the T-rex system (invitrogen), following manufacturer's instruction. Keywords: Genetic modification Overall design: Pulse-chase experiment, examining expression profile at 0h, 12h, 24h from Doxycycline addition. For details, please refer to our publication (American Journal of Physiology GI and Liver disease, e-pub at Nov 20th, 2008)
INSTRUMENT(S): Hitachisoft AceGene Human Oligo Chip 30K Subset A
Project description:The cell line was modified to express activated form of Notch1 (NICD1 of mouse origin) upon Doxycycline addition to the culture medium generated by T-rex system (invitrogen) following manufacturer's instruction. Keywords: Genetic modification Pulse-chase experiment, examining expression profile at 0h, 12h, 24h from Doxycycline addition. For details, please refer to our publication (American Journal of Physiology GI and Liver disease, e-pub at Nov 20th, 2008)
Project description:A human colon carcinoma-derived cell line LS174T was modified to overexpress Hes1, a bHLH-type transcription factor, upon doxycycline addition (designated as LS174T-tetON-Hes1 cells), using the T-rex system (Invitrogen). We have previously shown that these cells can overexpress Hes1 under the control of CMV promoter (Zheng et al, Inflamm Bowel Dis, 17;2251-2260, 2011), and the amont of the overexpressed Hes1 protein reaches to the maximal level in early as 3 hours from doxycycline addition (100ng/ml), which persists for up to 24 hours. In the present experiment, LS174T-tetON-Hes1 cells were treated with recombinant human IL-22 (20ng/ml) alone, doxycycline alone (100ng/ml), co-treated by both IL-22 and doxycycline, or left untreated (Control) for 24 hours, and subjected for analysis. Experiment was done using a modified sub-line of LS174T cell (LS174T-tetON-Hes1 cells), in which overexpression of Hes1 can be induced by a Doxycycline dependent manner. Four-condition experiment, Control vs. IL-22 stimulation, Control vs Doxycycline addition (Hes1 overexpression) and Control vs IL22 stimulation+Doxycycline addition (Hes1 overexpression). Biological replicates: One for each condition, independently grown and harvested. One replicate per array.
Project description:Notch signaling is widely implicated in mouse mammary gland development and tumorigenesis. To investigate the effects of acute activation of Notch signaling in the mammary epithelial compartment, we generated bi-transgenic MMTV-rtTA; TetO-NICD1 (MTB/TICNX) mice that conditionally express a constitutively active NOTCH1 intracellular domain (NICD1) construct in the mammary epithelium upon doxycycline administration. Overall design: Two timepoints (48h and 96h) of doxycycline induction in TetO-NICD1 (TICNX; control) and MMTV-rtTA; TetO-NICD1 (MTB/TICNX) mice with 3-4 replicates per timepoint
Project description:RNAi-seq of stable Flp-In™ T-Rex Hela cells that inducibly expressed PP1-NIPP1 and mutants. Overall design: Four cell types are investigated under two conditions in three independent experiments, resulting in 24 samples in total (i.e. 4x2x3). The four cell types are: (1) Flp-In™ T-Rex Hela cells with doxycycline-inducible EGFP transgene, (2) Flp-In™ T-Rex Hela cells with doxycycline-inducible EGFP-PP1-NIPP1 transgene, (3) Flp-In™ T-Rex Hela cells with doxycycline-inducible EGFP-PP1m-NIPP1 transgene (PP1 is mutated at Aa 64, i.e. D64A), (4) Flp-In™ T-Rex Hela cells with doxycycline-inducible EGFP-PP1-NIPP1m transgene (NIPP1 is mutated at the position AA 68-71, i.e. alanine mutation of NIPP1 residues 68-71). The two conditions are (1) not exposed to doxycycline, (2) treated with doxycycline. Three replicates are collected from three independent experiments.
Project description:Chronic activation of Notch1 synergizes with multiple oncogenic pathways altered in early prostate cancer to promote the development of prostate adenocarcinoma. Expression profiling revealed that these tumors display features of epithelial-to-mesenchymal transition, a cellular state associated with increased tumor aggressiveness.Tumors driven by Notch1 intracellular domain in combination with multiple pathways altered in prostate cancer are metastatic and resistant to androgen deprivation. Overall design: Mouse prostate tumors initiated by the combination of NICD1/kRasG12D, NICD1/myrAKT, or NICD/Myc and normal mouse prostates were subjected to high-throughput RNA-sequencing and differential gene-expression analysis
Project description:Airway basal cells (BC) function as progenitor cells capable of differentiating into ciliated and secretory cells to replenish the airway epithelium during physiological turnover and repair. The objective of this study was to define the role of Notch signaling in regulating human airway BC differentiation into a pseudostratified mucociliated epithelium. Notch inhibition with γ-secretase inhibitors demonstrated Notch activation is essential for BC differentiation into secre-tory cells and ciliated cells, but more so for the secretory lineage. Sustained Notch activation via lentivirus expression of the intracellular domain of each Notch receptor (NICD1-4) demonstrated that the Notch 2 and 4 pathways have little effect on BC differentiation, while activation of the Notch1 or 3 pathways has a major influence, with persistent expression of NICD1 or 3 resulting in a skewing toward secretory cell differentiation with a parallel decrease in ciliated cell differentiation. These observations provide insights into the control of the balance of BC differentiation into the secretory vs ciliated cell lineage, a balance that is critical for maintaining the normal function of the airway epithelium in barrier defense against the inhaled environment. Array-based expression profiling of the Notch signaling pathway genes specifically in human airway basal cells.
Project description:We explore the transcriptional response of mammalian cells undergoing various insults to Golgi homeostasis. HEK293 cells (Flp-In T-REx 293 cells) stably containing a doxycycline-inducible Golgi-localized HaloTag2 construct (GA-HT2) were treated with the ionophore nigericin, the glycosylation inhibitor xyloside, or were induced by doxycycline and treated with the hydrophobic tag HyT36 to induce destabilization of GA-HT2. We found that while nigericin and xyloside induce global transcriptional changes, destabilization of GA-HT2 induces a Golgi-specific response. Overall design: HEK293/GA-HT2 cells were treated in duplicate with nigericin, xyloside, HyT36, or the vehicle DMSO in the absence of doxycycline. In addition, cells were treated with DMSO or HyT36 in the presence of doxycyline.
Project description:Transcriptional profiling of murine splenic mature B cells that have experienced activation-induced cytidine deaminase (Aicda) gene expression comparing control untreated B cells with B cells in which transgenic Notch1 intracellular domain (NICD1) gene, coding for an active form of Notch1, began to be expressed after Aicda promoter-driven expression of Cre recombinase. B cells that have experienced Aicda promoter activation were visualized by Cre-inducible expression of red fluorescent protein (RFP) which was inserted in the Rosa26 locus. Total RNA was extracted from splenic CD19+ RFP+ B cells isolated from mice using an RNeasy Mini kit (Qiagen, Hilden, Germany). Equal amounts of total RNA derived from 3 control mice and 3 mice with conditional activation of Notch1 signaling in mature B cells were pooled and submitted for gene expression microarray analysis. Goal was to determine the effects of conditional activation of Notch1 signaling in mature B cells. Overall design: Two-condition experiment, mature B cells vs. Notch1-activated mature B cells. Biological replicates: Equal amounts of total RNA from 3 mice per group were pooled together.
Project description:In homeostasis of adult vertebrate tissues, stem cells are thought to self-renew by infrequent and asymmetric divisions that generate another stem cell daughter and a progenitor daughter cell committed to differentiate. This model is based largely on in vivo invertebrate or in vitro mammal studies. Here we examine the dynamic behaviour of adult hair follicle stem cells in their normal setting by employing mice with repressible H2B-GFP expression to track cell divisions and Cre inducible mice to perform long-term single cell lineage tracing. We provide direct evidence for the infrequent stem cell division model in intact tissue. Moreover, we find that differentiation of progenitor cells occurs at different times and tissue locations than self-renewal of stem cells. Distinct fates of differentiation or self-renewal are assigned to individual cells in a temporal-spatial manner. We propose that large clusters of tissue stem cells behave as populations, whose maintenance involves unidirectional daughter-cell fate decisions. We used microarrays to expression profile the stem cell progeny generated at distinct differentiation and self-renewal stages. Overall design: We employed double transgenic mice, K5tTA x pTRE-H2B-GFP in which an epithelial Keratin 5 (K5) promoter drove repressible histone H2B-GFP expression. Repression is achieved by feeding the mice doxycycline for a period of time (chase), when the H2B-GFP dilutes in cells by 2-fold at division. This allowed us to quantify precise proliferation history in vivo, from the amount of H2B-GFP fluorescence retained in cells after chase. After various chase periods we sacrificed mice at different ages corresponding to distinct phases of hair cycle. We isolated skin cell suspensions from these mice and stained them for surface expression of stem cell niche bulge(Bu) markers CD34 and alpha 6-integrin. Fluorescence-activated cell sorting (FACS) revealed histograms with distinct peaks of 2-fold median H2B-GFP intensity corresponding to distinct divisions. For microarray, we FACS sorted bulge(Bu) and non-bulge(NonBu) cells based cell surface marker staining and their proliferation history with H2B-GFP intensity, after two doxycycline chase schemes (Postnatal Day 18-PD21 or PD22-PD25), then extract total RNA from each cell fractions. For microarrays 5 ng of high quality RNA (Bioanalyzer, Agilent) were amplified (Ovation Amplification; Nugene) in the Cornell Microarray Core Facility. GeneChip IVT labelling was followed by Gene Chip MOE 430 2.0 hybridization, GeneArray 3000 scanning, GCOS generation of present calls and signal values (Affymetrix).
Project description:Pulse chase measurements using thiouracil (DTU) labeling via UPRT and chasing with uracil Data from tachyzoites is labeled "DTU Pulse Chase". Two independent pulse chase experiments were performed in tachyzoites, pulse chase 1 and 2. Duplicate arrays at each timepoint were performed for pulse chase 2 (2 a and b). Data from bradyzoites are labeled "DTU Bradyzoite Pulse Chase". Two independent pulse chase experiments were performed in bradyzoites and a single set of arrays were performed for each experiment. Just one chase timepoint was used in the bradyzoite experiments, the 2 hour chase.