Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RAW264.7 cells were treated with KDO, IFN beta and 8Br

ABSTRACT: RAW 264.7 cells were stimulated with KDO (100 ng/ml), IFN-β (300 pM) and/or 8-Br 100 μM. These ligands were applied individually or in combination with KDO for 60 min and 120 min. Total RNA was extracted using TriPure (Roche) following its protocol. Keywords: designed RAW 264.7 cells were stimulated with KDO (100 ng/ml), IFN-β (300 pM) and/or 8-Br 100 μM. These ligands were applied individually or in combination with KDO for 60 min and 120 min. Total RNA was extracted using TriPure (Roche) following its protocol. The detailed procedure is available at http://www.signaling-gateway.org/data/ProtocolLinks.html (AfCS Procedure Protocol PP00000009). Duplicate experiments were done for each treatment. Oligonucleotide array fabrication and annotation 16 K mouse oligonucleotide arrays were fabricated with 15,631 oligomers of 65-bp or 70-bp long. The oligomers were purchased from Operon and Sigma-Genosys and were inkjet-printed onto glass slides by Agilent Technologies. The list of genes is available through the GEO (Gene Expression Omnibus - http://www.ncbi.nlm.nih.gov/geo) as an accession number GPL254. Gene expression analysis The hybridization experiment and analysis were performed as previously described (Park et al., 2004; Zhu et al., 2006; Zhu et al., 2004). Cy5-labeled cRNA (RNA of ligand treated cells) and Cy3-labeled cRNA (RNA of time matched controls) were hybridized in the array. Dye-swap labeling was performed for each pair of samples. The arrays were scanned with Agilent Scanner G2505A (Agilent Technologies) and image files were extracted with background subtraction and dye-normalization using Agilent G2566AA Extraction Software version A.6.1.1 (Agilent Technologies).

ORGANISM(S): Mus musculus

SUBMITTER: Sangdun Choi

PROVIDER: E-GEOD-11449 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Overexpression of programmed cell death 5 gene (PDCD5) in tumor cells enhances apoptosis triggered by growth factor or serum deprivation, and overexpression of its homolog, OsPDCD5, induces the early death of transgenic plants. In this work, a system of inducible OsPDCD5 expression using a heat shock promoter was developed to study PCD in rice at different developmental stages.The results showed that in three-leaf aged and older seedlings, OsPDCD5 could independently induce PCD. In altered plants, OsPDCD5 expression caused lesion mimic phenotype, abnormal leaf morphology, nuclear condensation, DNA fragmentation, and H2O2 production. But two-leaf aged and younger seedlings seedlings showed no visibly morphological phenotype after OsPDCD5 expression, suggested that young seedlings possessed some mechanism inhibiting OsPDCD5 induced PCD. The transcripts of 24-day-old transgenic Zhonghua 11 seedlings heat-shocked for 6 h (before the appearance of PCD phenotypes) were compared with untreated seedlings using microarray analysis (3-Leaf Group) to study the genes involved in OsPDCD5 induced PCD. The experiment was also performed using 18-day-old seedlings to discover why OsPDCD5 expression did not induce PCD in very young seedlings (2-Leaf Group). RNA samples were extracted using TRIzol (Invitrogen) as described by the manufacturer. Microarray analyses were carried out using a competitive hybridization method using the Agilent microarray system. All procedures were carried out according to the manufacturer's protocols. Briefly, total RNA from each sample was used to synthesize cRNA and was labeled with cyanine-5 (Cy5)- or cyanine-3 (Cy3)-labeled CTP. The labeled cRNAs (including a heat-shocked and a control sample) were competitively hybridized to the Agilent Rice Oligo Microarrays, and then washed. The hybridized slides were scanned using Agilent DNA Microarray Scanner and data points were extracted using Agilent Feature Extraction software. Two comparisons were made between biologically independent samples.

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RAW264.7 cells were treated with KDO, IFN beta and 8Br

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