Secondary screening and association of genes to the ERK and/or GSK3 pathways.

<p>(A) Schematic representation of the strategy used to stratify the high confidence positive hits via either the ERK and/or GSK3 pathways (1i-screens) using the <i>rex1-GFP</i> reporter system. A counter screen was performed in the presence of the two kinase inhibitors (CHIR99021 and PD0325901)(“+2i”) and remaining hits were tested when either the GSK3 inhibitor (CHIR99021; -GSKi) or the MEK inhibitor (PD0325901; -MEKi) was withdrawn. The Venn diagram illustrates the four different hit categories. (B) Heatmap summary depicts the stratification of the hits according to their effects on the GFP(+)/GFP(−) ratio upon withdrawal of the GSK3 inhibitor (-GSK3i) or MEK inhibitor (-MEKi). The numbers of genes in each category are indicated. The colour scale represents the ratio of high to low GFP expressing cells for each siRNA pool in each of the “1i” screens. (C and D) Heatmaps of the enriched GO terms identified for genes corresponding to hits specific to the total dataset from the “2i” screen (274), or hits from the “1i” screens; ERK (133), or GSK3 (168) pathways. Each GO term is scored by −log₁₀(P-value). The associated GO term descriptions are indicated (the GO terms enriched in only the ERK or GSK3 categories are indicated in red or green font respectively). Distinct functional groups corresponding to terms associated with cell signaling (C) and gene expression (D) are manually clustered. (E) STRING network analysis of the core network formed by the 274 genes associated with signal-dependent loss of pluripotency and promoting early differentiation processes in the mouse embryonic stem cells. Genes are grouped according to common biological processes. The coloured lines of edges represent confidence scores of interconnectivity. Dark blue lines represent 0.8–1, light blue lines represent 0.6–0.8, and light grey lines represent 0.4–0.6 confidence levels, respectively.