Expression of genes involved in Ca²⁺ signaling in GICs correlating with a NSC-associated transcriptome.

<p>(A) GIC lines rank ordered in relation to NSC lines (second component in a principle component analysis of microarray based mRNA expression data from Pollard et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115698#pone.0115698-Pollard1" target="_blank">[11]</a>, where the first component segregates NSCs and GICs from normal brain tissue). GliNS1 is derived from the G144ED line in the Pollard et al study. (B) Re-analysis of transcriptome profiles in Pollard et al comparing GICs to NSCs indicating a NSC-proximal cluster of stem-like GICs with high similarity to NSCs, sharing e.g. SOX2 and BLBP expression. NSC-distal GIC lines in contrast expressed microglia markers, such as CXCL2, CXCL5 and CCL20. (C) De novo RNA sequencing analysis and pairwise comparisons of NSCs and three individual GIC lines (GliNS1, G179NS and G166NS) showed that NSCs expressed a larger number of genes with 10-fold higher gene expression compared to all GIC lines. (D) Pairwise comparisons of NSCs to the GIC lines GliNS1, G179NS and G166NS, individually. Gene enrichment and gene ontology analysis of sequencing based transcriptome profiles, identified an enrichment of Ca²⁺ signaling genes in NSCs, which increased with rank order distal to NSC in pairwise comparisons. (E) Pairwise comparisons of the NSC-proximal (GliNS1) and NSC-distal (G166NS) GICs. Gene enrichment and gene ontology analysis suggested a switch in Ca²⁺ permeable channels to Ca²⁺ binding genes in the NSC-distal GIC line (upper boxes). In volcano plot, gene names in green denote ion channel/pump/transporter related genes, whereas gene names in purple denote Ca²⁺ binding proteins genes. The volcano plot of the comparison of NSC-proximal and NSC-distal GICs revealed a larger number of ion channels expressed in the NSC-proximal GIC (GliNS1).</p

Transcriptome analysis of drug response in GliNS1 and G166NS.

<p>Transcriptional response to increased cytosolic Ca²⁺ (A23187), was investigated by RNA sequencing after 7 hours of drug exposure in the NSC-proximal GIC line GliiNS1 and the NSC-distal line G166NS. Volcano plots of significantly (p<0.05) altered gene expression in GliNS1 (A) and G166NS (C) with shared induced genes marked in red and green (Ca²⁺ activated transcription factor NFATC2). Note the differences in x-axis indicating higher all global induction of gene expression in GliNS1. (B) Gene enrichment and gene ontology analysis of genes with a significant change in expression (p<0.05) in GliNS1, identified genes involved in cell cycle progression as well as ER/golgi associated functions and cellular stress response. (D) Gene enrichment analysis of genes downregulated at least 3-fold in GliNS1 and upregulated at least 1.5-fold in G166NS.</p

Gene expression correlating with high Ca²⁺ sensitivity in 9 GIC lines.

<p>(A) A correlation analysis of genome wide mRNA expression (microarray analysis) and sensitivity to Thapsigargin (1 uM) in 9 additional GIC lines, retrieved 785 genes correlating with Ca²⁺ drug sensitivity. Gene enrichment and ontology analyses identified involvement of genes affecting proliferation, oxygen and RNA metabolism, catabolism and Ca²⁺-mediated signaling. (B) 385 genes positively correlating with high sensitivity were filtered first for genes also expressed higher in the NSC-proximal GIC line GliNS1 and thereafter also being downregulated in this line upon differentiation, which was found to reduce Ca²⁺ drug sensitivity, retrieving a set of nine genes, including the AMPA receptor coding GRIA1.</p