Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of mouse embyronic stem cells cultured with PI3-K signalling inhibitor LY294002 for different lengths of time to identify PI3K-target genes

ABSTRACT: This experiment is part of the FunGenES project (FunGenES - Functional Genomics in Embryonic Stem Cells partially funded by the 6th Framework Programme of the European Union, http://www.fungenes.org).The experiment was conducted at the Unyversity of Bath, Bath, UK. Aim is to identify genes regulated by PI3K-dependent signaling in undifferentiated ES cells. Materials and methods: E14tg2a cell line was used. Cells were cultured in Knock-out DMEM supplemented with 15% (v/v) Knock-out serum replacement, 1000U/ml LIF, 2mM glutamine, 50 ?M 2-mercaptothanol and 1x NEAA (as described in Handbook of work methods, 4.3.1.1). Cells were plated at 5x105 per 10cm gelatin-coated TC dish for 24h. ES cells were then starved for 24h in media lacking LIF and pre-incubated for 30 minutes with 5 M LY294002 (the concentration of this PI3K inhibitor that we have shown to perturb self-renewal in the presence of LIF and which knocks down PI3K signals) or DMSO alone. Cells were then stimulated with 103U/ml mLIF (Chemicon) for 2h (which gave maximal activation of STAT3), 24h, 48h or 72h.

Relationships between samples

Samples were either maintained in LIF or LIF plus LY294002 (PI3K inhibitor) for the required treatment times.

Treatments

Treatment times with PI3K inhibitor LY294002 at 5 M <96> 2h (n=5), 24h (n=3), 48h (n=3), 72h (n=3). DMSO was used as vehicle alone treatment control.

Culture conditions

Gelatin-coated TC dishes (Nunc)

RNA isolation method

RNA Easy with on-column DNase treatment

Experimental description

This information is included above. Briefly, E14tg2a ES cells were either maintained in LIF or LIF plus LY294002 (5 M) for 2, 24, 48 or 72 hours, after which RNA was extracted from each sample and sent for expression profiling. During this time-course, only 72h time points plus LY294002 showed phenotypic evidence of differentiation.

ORGANISM(S): Mus musculus

SUBMITTER: Oliver Hummel

PROVIDER: E-TABM-674 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Characterization of the phosphoinositide 3-kinase-dependent transcriptome in murine embryonic stem cells: identification of novel regulators of pluripotency.

Storm Michael P MP Kumpfmueller Benjamin B Thompson Belinda B Kolde Raivo R Vilo Jaak J Hummel Oliver O Schulz Herbert H Welham Melanie J MJ

Stem cells (Dayton, Ohio) 20090401 4

Phosphoinositide 3-kinase (PI3K)-dependent signaling has been implicated in the regulation of embryonic stem (ES) cell fate. To gain further insight into the mechanisms regulated by PI3Ks in murine ES cells, we have performed expression profiling using Affymetrix GeneChips to characterize the transcriptional changes that arise as a result of inhibition of PI3K-dependent signaling. Using filtering of greater than 1.5-fold change in expression and an analysis of variance significance level of p < ...[more]

PMID: 19350676