title: Identification of LMX1B target genes in a tet-off inducible HeLa cell line and in the mouse kidney

status: Public on Dec 04 2008

city: Regensburg

first: Anne

last: Rascle

address: Universitaetsstrasse

institute: University of Regensburg

summary: LMX1B is a LIM-homeodomain transcription factor essential for development. Putative LMX1B target genes have been identified through mouse gene targeting studies; however, in the absence of in vivo molecular characterization of their regulation, their identity as direct LMX1B targets remains hypothetical. We describe here the first molecular characterization of LMX1B target gene regulation. A tetracycline-inducible expression system and microarray analysis showed that a subset of NF-kappa B target genes, including IL-6 and IL-8 are upregulated in LMX1B-expressing HeLa cells. Chromatin immunoprecipitation assays revealed that LMX1B binds to the proximal promoter region of IL-6 and IL-8 in vivo, in the vicinity of the characterized kappa B site, and that LMX1B recruitment correlates with an increased NF-kappa B DNA association. Inhibition of NF-kappa B activity by short interfering RNA-mediated knock-down of p65 impairs LMX1B-dependent induction of NF-kappa B target genes, while activation of NF-kappa B activity by TNF-alpha results in a synergistic induction of these genes by LMX1B. IL-6 promoter-driven reporter assays showed that the kappa B site and an adjacent putative LMX1B binding motif are both involved in LMX1B-mediated transcription. Expression of a number of NF-kappa B target genes is affected in the kidney of Lmx1b-/- knock-out mice, thus supporting the biological relevance of the data obtained in the human cell line. Together, these data demonstrate for the first time that LMX1B directly regulates transcription of a subset of NF-kappa B target genes in cooperation with nuclear p50/p65 NF-kappa B.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design:
Human subset (GSM303547-GSM303550): Two biological replicates of non-expressing HtTA-LMX1B cells (grown in doxycycline-containing medium) and of LMX1B-expressing HtTA-LMX1B cells (grown for 4 days in doxycycline-free medium) were processed for gene expression array analyses using an Affymetrix platform.

Mouse subset (GSM304379-GSM304384): Three kidney samples from newborn wild-type mice and from newborn Lmx1b-/- knock-out mice were processed for gene expression array analyses using an Agilent platform.

first: Tanja

last: Neumann

first: Anne-Sarah

last: Raschta

first: Astrid

first: Eva

last: Heining

first: Juergen

last: Kastner

first: Christoph

last: Moehle

first: Thomas

last: Stempfl

first: Ralph

last: Witzgall


title: Knock-out of the nuclear localization in pp65 protein of Cytomegalovirus:  biologic and immunologic effects

status: Public on Jan 31 2009

city: Duarte

first: Xiwei

last: Wu

state: CA

address: 1500 E. Duarte Rd.

department: Computational and Quantitative Medicine

country: USA

institute: City of Hope National Medical Center

summary:
The CMVpp65 protein contains 2 bipartite nuclear localization signals (NLS) at 415-438aa and 537-561aa near the carboxy terminus of CMVpp65 and a phosphate binding site related to kinase activity at lysine-436.  A mutation of pp65 having K436N (CMVpp65mII) and further deletion of aa537-561 resulted in a novel protein (pp65mIINLSKO) that is kinase-less and has markedly reduced nuclear localization.  The purpose of this report was to study the biologic characterization of this protein and its immunogenicity compared to native pp65.Using RNA microarray analysis, expression of the CMVpp65mIINLSKO had less effect on cell cycle pathways than did the native CMVpp65 and a greater effect on cell surface signalling pathways involving immune activity.  It is concluded that the removal of the primary NLS motif from pp65 does not impair its immunogenicity and may actually be advantageous in the design of a vaccine.

Keywords: comparison of cellular response to wild-type gene expression vs a mutated gene.

overall_design: The MRC-5 cells were transduced in triplicate, with rAAVintA,  rAAVpp65N, rAAVpp65NLSKO for 48hrs and the total RNA was purified with RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. One µg of each sample was processed using the Affymetrix GeneChip Whole Transcript Sense Target Labeling Assay.  After ribosomal RNA reduction (Invitrogen, Carlsbad, CA) to remove most of the 18S and 28S rRNAs, the GeneChip WT (whole transcript) cDNA Synthesis Kit, WT cDNA Amplification Kit, and WT Terminal Labeling Kit (Affymetrix, Inc., Santa Clara, CA) were used for target preparation. Ten µg of cRNA (antisense RNA) were added to the second-cycle cDNA reaction followed by fragmentation and Terminal Labeling process.  The hybridization cocktails containing 2.5 ug of the fragmented, end-labeled cDNA were applied to the GeneChip® Human Gene 1.0 ST arrays. The Genechip® Human Gene 1.0 ST array was chosen because it offers the whole transcript coverage consisting of 28,869 genes on the array by approximately 26 probes spread across the full length of the gene.

first: John

middle: A

last: Zaia

first: Xiuli

last: Li

middle: E

last: Franck

first: Lia

last: Thao

first: Ghislaine

last: Gallez-Hawkins


title: Effect of 4-Hydroxynonenal on gene expression. Comparison of HSF1-siRNA silenced vs control cells

status: Public on Oct 01 2008

city: Nashville

first: Aaron

middle: Thomas

last: Jacobs

state: TN

address: 23rd Ave. @ Pierce Ave.

department: Biochemistry

institute: Vanderbilt University

summary: 4-Hydroxynonenal (HNE), a cytotoxic and diffusible electrophile generated by the spontaneous decomposition of oxidized lipids, has a suspected role in neurodegenerative and inflammatory disease processes. In addition to promoting cell death, elevated levels of HNE lead to the engagement of cytoprotective signaling pathways, including the heat shock, antioxidant, DNA damage, and ER stress responses. Activation of the heat shock response, mediated by the transcription factor heat shock factor 1 (HSF1), is critical for maintaining cellular viability in the presence of HNE. Accordingly, silencing HSF1 expression using siRNA enhances the toxicity of HNE. Microarray analysis of samples from control and HSF1-silenced cells was performed to investigate which associated changes in gene could be responsible for the decrease in cellular viability.

overall_design: Four different experimental conditions were investigated. Samples were prepared and analyzed in triplicate. Samples 1-3 received NEG control siRNA and DMSO (vehicle) for 6 h. Samples 4-6 received NEG control siRNA and 50 uM HNE for 6 h. Samples 7-9 received HSF1 siRNA and DMSO (vehicle) for 6 h. Samples 10-12 received HSF1 siRNA and 50 uM HNE for 6 h.

middle: T

first: Lawrence

middle: J

last: Marnett


title: Regulation of endothelial gene expression by miR-126 in human and zebrafish

status: Public on Aug 12 2008

city: San Francisco

first: Jason

middle: E.

last: Fish

address: 1650 Owens Street

department: Gladstone Institute of Cardiovascular Disease

institute: J. David Gladstone Institute

summary: This SuperSeries is composed of the SubSeries listed below.

sample_organism:
Danio rerio
Homo sapiens

platform_organism:
Danio rerio
Homo sapiens

overall_design: Refer to individual Series


title: Gene expression profiling by DNA microarray in Colo741 cell line transfected by KRas-G12D or KRas-G12V

status: Public on Dec 18 2008

city: Genova (GE)

first: Massimiliano

last: Monticone

address: Largo R. Benzi, 10

country: Italy

institute: IRCCS AOU San Martino – IST, Genova

summary:
Mutations of the proto-oncogene KRas, together with the inactivation of the onco-suppressor genes APC and TP53, are considered to have an important role both in the carcinogenesis and in the colorectal tumor progression.
The oncogene K-ras has activating mutations, especially in codon 12, in about 40% to 50% of colorectal carcinomas (Andreyev et al., 1998). The mechanism by which oncogenic K-ras alone contribuites to tumor progression in colon cancer is largely unknown. To this end, we have developed a model system where the colorectal adenocarcinoma cell line Colo741 has been stably trasfected with the Kras-G12V or Kras-G12D mutated oncogenes.
Our results demonstrate how expression profiling data can be used to interpret changes in cellular characteristics induced by transfection and hence provide some clues as to the mechanisms by which different activated K-Ras impair processes mediated by endogenous wild-type KRas proteins.

overall_design:
We made for the production of cell clones that could be utilized as useful in vitro models. The adenocarcinoma cell line Colo741 has been chosen to produce stable transfectants for two mutant forms of KRas2 (Gly12Val and Gly12Asp) and the wild-type coding sequence (as experiment control). After the transfection, the cells have been selected (with G418), cloned (by limited dilutions) and screened (by RT-PCR). The total RNAs, Dnase-treated, from five cell clones per condition, have been pooled.

The samples, constituted by biotin-labeled cRNA, derived from a pool of five cell clones obtained from transfection of the adenocarcinoma cell line Colo741 with either pcDNA3.1-KRas2(Wt) or pcDNA3.1-KRas2(Gly12Val) or pcDNA3.1KRas2(Gly12Asp). They were hybridized to a Human Gene 1.0 ST array (Affymetrix). Two technical replicates have been performed for each condition.

first: Emanuela

last: Biollo

first: Francesco

last: Romeo

first: Walter

last: Giaretti

first: Patrizio

last: Castagnola


title: Regulation of human endothelial gene expression by miR-126

summary:
Fish, JE, Santoro, MM, Morton, SU, Yu, S, Yeh, RF, Wythe, JD, Ivey, KI, Bruneau, BG, Stainier, DYR, and Srivastava, D. (2008). miR-126 Regulates Angiogenic Signaling and Vascular Integrity. Developmental Cell 15, 272-284.

Precise regulation of the formation, maintenance, and remodeling of the vasculature is required for normal development, tissue response to injury, and tumor progression. How specific microRNAs intersect with and modulate angiogenic signaling cascades is unknown. Here, we identified microRNAs that were enriched in endothelial cells derived from mouse embryonic stem (ES) cells and in developing mouse embryos. We found that miR-126 regulated the response of endothelial cells to VEGF. Additionally, knockdown of miR-126 in zebrafish resulted in loss of vascular integrity and hemorrhage during embryonic development. miR-126 functioned in part by directly repressing negative regulators of the VEGF pathway, including the Sprouty-related protein SPRED1 and phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2/p85-β). Increased expression of Spred1 or inhibition of VEGF signaling in zebrafish resulted in defects similar to miR-126 knockdown. These findings illustrate that a single miRNA can regulate vascular integrity and angiogenesis, providing a new target for modulating vascular formation and function.

overall_design: Human umbilical vein endothelial cells, which express high levels of miR-126, were utilized to identify mRNA targets of miR-126.  Cells were electroporated with 15 nmol of an antisense morpholino that blocks the processing of the miR-126 pri-cursor.  A non-targeting morpholino (15 nmol) was used as a control.  RNA was isolated 72 hr following electroporation and arrays were performed using Affymetrix Human Gene 1.0 ST arrays.  Analysis was performed on three biological replicates of control morpholino and miR-126 antisense morpholino transfected cells

first: Ru-Fang

last: Yeh

first: Deepak

last: Srivastava


title: Gene expression change in the liver of tumor bearing mice by TSU68 treatment

status: Public on Jun 18 2008

city: Hamamatsu

first: M

last: Yamamoto

address: 1-20-1 Handayama, Higashiku

department: 2nd Dept. Surgery

country: Japan

institute: Hamamatsu Univ. Sch. of Med.

summary: The aim of this study was to investigate the inhibitory effect of TSU68, a tyrosine kinase inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2), platelet-derived growth factor receptor beta (PDGFRβ) and fibroblast growth factor receptor 1 (FGFR1), on colon cancer liver metastasis and to test the hypothesis that TSU68 modulates the microenvironment in the liver before the formation of metastasis.

overall_design:
The human colon cancer TK-4 was implanted orthotopically into cecal walls of 6-week-old male BALB/c nu/nu mice (Clea Japan, Tokyo, Japan). The animals were treated with TSU68 (400 mg/kg/day, twice-daily, p.o.) or vehicle from 7 days after orthotopic implantation. After one week of drug administration, livers were removed and total RNA was extracted.
We established four different groups of mice; non-tumor-bearing and treated with vehicle alone (NT-Co), non-tumor-bearing and treated with TSU68 (NT-TSU), tumor-bearing and treated with vehicle (T-Co), and tumor-bearing and treated with TSU68 (T-TSU). NT-Co, T-Co and T-TSU group were applied to microarray analysis.

first: Masayoshi

first: Hirotoshi

last: Kikuchi

first: Manabu

last: Ohta

first: Takashi

last: Uehara

first: Tomohiko

last: Setoguchi

first: Toshiki

last: Kawabata

first: Yoshihiro

last: Hiramatsu

first: Kenji

last: Kondo

first: Megumi

last: Baba

first: Kinji

last: Kamiya

first: Tatsuo

last: Tanaka

first: Masatoshi

last: Kitagawa

first: Hiroyuki

last: Konno


title: Genes activated by s.a through the activity of phosphatidylinositol 4-kinase- Role of phosphatidylinositol 4-kinase

status: Public on Apr 12 2007

city: evry

first: taconnat

last: Ludivine

address: 2 rue gaston crémieux

department: URGV

country: France

institute: INRA

summary:
Cell suspension culture of Arabidopsis thaliana (ecotype Columbia) was treated by 250 uM salicylic acid and by two different concentrations of wortmannin (1 uM and 30 uM) for 4 hours.
Keywords: dose response,treated vs untreated comparison

overall_design: 10 dye-swap - CATMA arrays

first: Ludivine

last: Taconnat

first: Jean-Pierre

middle: P

last: Renou

first: Eric

last: Ruelland

first: Alain

last: Zachowski

first: Marie-Laure

middle: L

last: Martin-Magniette


title: Chick myopia

status: Public on Jul 26 2007

city: Philadelphia

first: Alice

middle: M

last: McGlinn

state: PA

address: 602D Richards Building 3700 Hamilton Walk

department: Ophthalmology

institute: University of Pennsylvania School of Medicine

summary:
One-day old white Leghorn chicks were housed in brooders with a 12 hr light:dark cycle, using General Electric chroma 50 fluorescent lighting with irradiance of approximately 50μW/cm2 at chick eye level. They received Purina Chick Chow food and water ad libitum. At one week of age and at the onset of the light phase, the chicks were anesthetized with inhalation ether, and a unilateral translucent white plastic goggle was glued to the periorbital feathers to induce ipsilateral form-deprivation myopia, alternating between the left or right eye.
After either 6 hrs (n=8) or 3 days (n=8) of goggle wear, the chicks were killed by decapitation. The enucleated eyes were opened at the equator, and the retina/RPE was dissected together from both the goggled and control eyes. The tissues were individually frozen and stored in liquid nitrogen until processed
Keywords: Gene expression

overall_design: Six biological replicates used for each: 3 day control retina, 3 day experimental, 6 hour control retina, and 6 hour experimental.

first: Donald

last: Baldwin

first: John

middle: W

last: Tobias

first: Murat

last: Budak

first: Tejvir

middle: S

last: Khurana

first: Richard

last: Stone


title: Microarray comparison of RNAi chc101 and mbd101 transgenic plants and non transgenic siblings after UV-B exposure

status: Public on Dec 31 2008

city: Rosario

first: Paula

last: Casati

state: Santa Fe

address: Suipacha 531

department: Biological Sciences

country: Argentina

institute: Universidad Nacional de Rosario

summary:
Mutant maize lines expressing RNAi gene silencing constructs directed to two distinct classes of chromatin associated proteins show heightened sensitivity to UV-B including sunburned necrosis and down regulation of chromatin genes (Casati and Walbot, 2006).  We have available transgenic maize expressing an RNAi for these chromatin factors: a subunit of a chromatin remodeling complex R, which is a SWI2/SNF2-like protein containing a SNF2 N and a helicase C domain (CHC101, AI746001); and a methyl-CpG-binding protein (MDB101, AI737448).
To better understand the processes that involve chromatin remodeling proteins in UV-B responses, we conducted transcriptome analysis of chromatin remodeling knockdown plants in under two radiation regimes:  plants were illuminated with UV-B lamps using fixtures mounted 30 cm above the plants (Phillips, F40UVB 40 W and TL 20 W/12) for 8 h; leaf samples were collected immediately after the radiation treatment. The bulbs were covered with CA filters to exclude wavelengths lower than 280 nm (UV-B). As a control, the bulbs were covered with PE filters to exclude wavelengths lower than 320 nm (minus UV-B).  We analyzed genes that are changed upon UV-B irradiation in the transgenic plants and compared to the responses in non transgenic plants. To differentiate between differences due to background specific genes expression, we used non-transgenic sibling as controls. For these experiments, we used Long oligonucleotides arrays from Agilent Technologies.  These studies will help to dilucidate how alteration in chromatin remodeling protein proteins affect gene expression, and the transcriptome analysis will help to understand why transgenic plants are hypersensity to UV-B.

Keywords: genetic modification-stress response

overall_design: Array hybridizations were carried out according to the manufacturer's instructions. For the experiments, Agilent Technologies microarrays were used, they contained 43,451 maize 60-mer probes and each slide had the same pattern of probes in quadruplicate (4x44 version), that were used to hybridize independent biological samples. Specifically, each array was hybridized with two samples, each of 0.825 μg labeled target cRNA, for 17 hours at 60°C. The samples were hybridized in a double loop design arrangement, as described by Kerr and Churchill et (2001). Data were acquired with an Agilent G2565BA scanner. Hybridizations for all 12 comparisons were highly correlated as assessed from correlations between median signal intensities (r2 = 0.97 for both dyes; data not shown) and between log2 ratios of the signals (r2 = 0.94; data not shown). Each sample was analyzed using 4 different biological replicates, and were hybridize to different arrays.

first: Virginia

last: Walbot


title: High temporal resolution profiling of U2OS fibroblasts

status: Public on Dec 15 2008

first: Michael

last: Hughes

address: 421 Curie Bvd, Brb 835

institute: UPenn

summary:
High-temporal resolution profiling was performed on U2OS fibroblasts to detect rhythmic transcripts

Keywords: time course

overall_design: Samples were collected every hour for 48 hours from dexamethasone-synchronized U2OS cells.  Cells synchronized with dexamethasone for 24 hours, and harvested from the 24th to 71st hour.  Samples were analyzed using Affymetrix arrays.

last: Hogenesch

first: Julie

last: Baggs


title: DKK2 Mediates Osteolysis, Invasiveness, and Metastatic Spread in Ewing Sarcoma

status: Public on Dec 21 2012

city: München

first: Olivia

last: Prazeres da Costa

state: Bavaria

address: Trogerstr. 30

department: Medical Microbiology, Immunology and Hygiene

country: Germany

institute: Technische Universität München

summary:
Ewing sarcoma, an osteolytic malignancy that mainly affects children and young adults, is characterized
by early metastasis to lung and bone. In this study, we identified the pro-metastatic gene DKK2
as a highly overexpressed gene in Ewing sarcoma compared with corresponding normal tissues. Using RNA
interference, we showed that DKK2 was critical for malignant cell outgrowth in vitro and in an orthotopic
xenograft mouse model in vivo. Analysis of invasion potential in both settings revealed a strong correlation of
DKK2 expression to Ewing sarcoma invasiveness that may be mediated by the DKK effector matrix
metalloproteinase 1 (MMP1). Furthermore, gene expression analyses established the ability of DKK2 to
differentially regulate genes such as CXCR4, PTHrP, RUNX2, and TGFb1 that are associated with homing,
invasion, and growth of cancer cells in bone tissue as well as genes important for osteolysis, including HIF1a,
JAG1, IL6, and VEGF. DKK2 promoted bone infiltration and osteolysis in vivo and further analyses defined
DKK2 as a key factor in osteotropic malignancy. Interestingly, in Ewing sarcoma cells, DKK2 suppression
simultaneously increased the potential for neuronal differentiation while decreasing chondrogenic and
osteogenic differentiation. Our results provide strong evidence that DKK2 is a key player in Ewing
sarcoma invasion and osteolysis and also in the differential phenotype of Ewing sarcoma cells.

overall_design: 6 samples (3x A673 cells; 3x SK-N-MC cells); for each cell line one sample was transfected with control non silencing siRNA and one sample with  DKK2 siRNA (siDKK2_1) and one sample with ITM2A siRNA (siITM2A_7) as an independent knock down.

first: Kristina

last: Hauer

first: Julia

last: Calzada-Wack

first: Thomas

middle: G

last: Grünewald

first: Stephanie

last: Plehm

first: Irene

last: Esposito

first: Thorsten

last: Buch

first: Stefan

last: Burdach

first: Günther

middle: H

last: Richter

first: Katja

last: Steiger

first: Daniel

last: Baumhoer


title: Role of phospholipase D in salycilic acid signaling in Arabidopsis thaliana suspension cells

status: Public on Nov 29 2007

city: Evry Cedex

first: Agnes

last: Yu

address: 2 rue Gaston Cremieux CP 5708

summary:
Our goal is to identify Salycilic Acid responsive genes dependent on PLD activation. - The experiment is done on Arabidopsis suspension cells, ecotype Columbia : Research of phospholipase D (PLD) activity implication in the response to SA. The use of primary alcohol, like N-butanol, makes possible to derive PLD activity towards the production of phosphatidylalcohol with the detriment of phosphatidic acid, the product of the PLD. Thus, in the presence of n-butanol, the response of the genes to SA dependent on phosphatidic acid will see their response disturbed. On the contrary, in the presence of tertiary butanol, the response of the genes should not be disturbed, tertiary alcohols not acting on the PLD.
Keywords: treated vs untreated comparison

overall_design:
14 dye-swap - CATMA arrays

Project coordinator: Eric Ruelland
Physiologie Cellulaire et Moléculaire des Plantes
UMR7180 CNRS/UPMC
3, rue Galilée
Le Raphaël
94200 Ivry sur Seine
email: eruellan@snv.jussieu.fr

first: Jean-Pierre

last: Renou

first: Eric

last: Ruelland

first: Marie-Laure

last: Martin-Magniette


title: Restoration of miR-214 expression reduces cell growth of myeloma cells through a positive regulation of P53 and inhibition of DNA replication

status: Public on Dec 28 2012

city: Salamanca

first: Norma

middle: Carmen

last: Gutierrez

department: Hematology

country: Spain

institute: University Hospital

summary: MicroRNAs have been demonstrated to be deregulated in multiple myeloma (MM). We have previously reported the downregulation of miR-214 in MM compared to normal plasma cells. In the present study, we have explored the functional role of miR-214 in myeloma pathogenesis. Ectopic expression of miR-214 reduced cell growth and induced apoptosis of myeloma cells. In order to identify the potential direct target genes of miR-214 which could be involved in the biological pathways regulated by this miRNA, gene expression profiling of H929 myeloma cell line transfected with precursor miR-214 was carried out. Functional analysis revealed significant enrichment for DNA replication, cell cycle phase and DNA binding. We show that miR-214 directly down-regulates the expression of PSMD10, which encodes the oncoprotein gankyrin, and ASF1B, a histone chaperone required for DNA replication, by binding to their 3'-UTR. In addition, gankyrin inhibition induced an increase of P53 mRNA levels and subsequent up-regulation in CDKN1A (p21Waf1/Cip1) and BAX transcripts, which are direct transcriptional targets of p53. In conclusion, we demonstrate that miR-214 function as a tumor suppressor in myeloma by a positive regulation of p53 and inhibition of DNA replication.

overall_design: H929 cell line was transfected with Pre-miR™ miRNA precursors pre-miR-214 or pre-miR™ miRNA negative, non-targeting control#1 (Ambion) at 50 nM concentration, using the nucleofector II system with C-16 program (Amaxa). The experiments were performed in triplicates.

first: Irena

last: Misiewicz-Krzeminska

first: María

last: Sarasquete

first: Dalia

last: Quwaider

first: Patryk

last: Krzeminski

first: Fany

middle: V

last: Ticona

first: Teresa

last: Paino

first: Manuel

last: Delgado

first: Andrea

last: Aires

first: Enrique

last: Ocio

first: Ramon

last: Garcia-Sanz

first: Jesus

middle: F

last: San Miguel

middle: C