Context-Specific Notch Targets Figure 11. Expression of Notch1 target genes is altered in RBP-J deficient ES cells. mRNA levels were measured by qPCR in ES cells cultured in self renewal medium (ESCs). The error bars indicate the standard deviation from 3 independent experiments. (A) Expression of potentially direct Notch1 target genes is increased in RBP-J deficient ES cells. Fold expression in relation to the expression of RBP-J +/+ D3 cells is shown. (B) The increased expression of Notch1 target genes in RBP-J -/- ES cells is repressed by exogenous RBP-J expression and further increased by a transcriptionally active form of RBP-J (RBP-J-VP16). Fold expression in relation to the expression of cells transfected with the control vector is shown. doi:10.1371/journal.pone.0011481.g011 (http://dchip.org). Microarrays were normalized to an array with median overall intensity (‘Control Mesoderm +OHT’) using the Invariant Set Normalization method [62]. Uninduced and OHT- induced samples of the same cell populations were compared. A gene was assigned as differentially regulated by Notch1, when it fulfilled the following criteria: (i) at least 2-fold up- or down- regulation, (ii) at least the higher expression value has to be judged as ‘present’ by the software, (iii) the absolute difference between induced/not-induced is higher than 50 (about 2-fold the mean background level of the ‘absent’ genes). For further analyses, redundant genes were removed as far as possible resulting in 465 genes and transcripts for all culture conditions applied. Data sets were deposited in NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE15268. Relative expression was visualized via heatmap using the MeV 4.2 of the TM4 software suite (http://www.tm4.org/; [63]). Values were Log2 transformed and normalized within each gene. Furthermore, genes were classified into Gene Ontology [64] or PANTHER groups [65] using the DAVID functional annotation analysis tool (http://david.abcc.ncifcrf.gov/). The GO and PANTHER terms used were: GOTERM_BP_ALL: GO:0006350,transcription OR PANTHER_MF_ALL: MF00036: Transcription factor (‘Transcription and TFs’); GOTERM_BP_ALL: GO:0006915,apoptosis; GOTERM_BP_ALL: GO:0030154,cell differentiation; GOTERM_BP_ALL: GO:0007049,cell cycle; GOTERM_BP_ALL: GO:0007154,cell communication; GO- TERM_BP_ALL: GO:0007155,cell adhesion; GOTERM_BP_ ALL: GO:0007242,intracellular signaling cascade; GOTERM_BP_ ALL: GO:0019222,regulation of metabolic process; GOTERM_ CC_ALL: GO:0005856,cytoskeleton; GOTERM_BP_ALL: GO: 0008283,cell proliferation; PANTHER_BP_ALL: BP00248: Mesoderm development. Genes of the groups were reassigned to their induction values and visualized as a heatmap with the MeV 4.2 software. Real-time RT-PCR Relative expression levels of 30 potential Notch1 target genes and Oct4 as a control were determined by real-time PCR on a 7900HT Fast Real-Time PCR System (Applied Biosystems) in 384-well PCR-plates (ABgene) using the TaqMan Gene Expres- PLoS ONE | www.plosone.org 15 July 2010 | Volume 5 | Issue 7 | e11481