(A) Flow cytometric recordings of DiBAC₄(3) loaded mESCs (n=10 000 cells, N=3) treated with 10 µM clofilium for 1 h. (B) Cell viability of mESCs treated with E4031 with and without sucrose (20 mM) or ouabain (1 µM) for 24 h. Data presented as mean ± SEM (n=4), one-way ANOVA, Tukey’ post-hoc test. (C) Images from time-lapse movies of mESCs treated with Erg inhibitor, E4031 (10 µM) and with the Na⁺,K⁺-ATPase inhibitor ouabain (1 µM).

<p>(A) Flow cytometric recordings of DiBAC₄(3) loaded mESCs (n=10 000 cells, N=3) treated with 10 µM clofilium for 1 h. (B) Cell viability of mESCs treated with E4031 with and without sucrose (20 mM) or ouabain (1 µM) for 24 h. Data presented as mean ± SEM (n=4), one-way ANOVA, Tukey’ post-hoc test. (C) Images from time-lapse movies of mESCs treated with Erg inhibitor, E4031 (10 µM) and with the Na⁺,K⁺-ATPase inhibitor ouabain (1 µM).</p

K⁺ permeability via Erg channel activity is critical for cell volume homeostasis.

<p>The Na, K-ATPase and K⁺ channels cooperate to establish a K⁺ ion circuit controlling intracellular [K⁺]. Inhibition of Erg channels lead to altered equilibrium between osmotic pressure and cortical actomyosin function resulting in an increase in cell volume ending in cell bursting. This could be counteracted by increasing extracellular osmolarity with sucrose or by blocking the influx of K⁺ ions by inhibiting the Na, K-ATPase with ouabain.</p

Erg inhibition decreased stiffness in mESCs.

<p>(A) A representative image of the cantilever placed over a mESC during atomic force microscopy. (B) In control conditions, blebbing cells showed a trend towards higher stiffness than non-blebbing cells (n=7, p=0.17, median indicated in box) and control cells that were subjected for hypertonic medium (sucrose 20 mM) for one hour showed reduced stiffness (n=11, p<0.001). (C) After 7 h of Erg inhibition (E4031; 10 µM) treated cells (n=8) were significantly (p=0.02, t-test unequal variance) less stiff than control cells (n=18).</p

Cell cycle dependent Erg1 channel expression.

<p>(A) Heat-map displaying mRNA differential expression of selected K⁺ channels in different cell cycle phases (ANOVA-test p<0.005). (B) Erg1 mRNA expression level evaluated using real-time PCR in cell cycle sorted mESCs (ANOVA-test). (C) Flow cytometry plot of live mESCs stained for an extracellular epitope of Erg1 channel. (D) Confocal images of mESCs sorted in different cell cycle stages and immunostained for Erg1 protein with cross section histograms (note that measurements across nucleoli were avoided) showing Erg1 immunostaining intensity with increased plasma membrane localization in G1.</p

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

Inhibition of Erg activity results in a mainly apoptosis independent cell death in G1 and early S phase.

<p>(A) mESCs were exposed to cisapride (10 µM) or vehicle for 6 h and DNA content was assayed using propidium iodide labeling by flow cytometry and quantified in respective cell cycle stages (one-way ANOVA, * p<0.05, ** p<0.01). (B) mESCs were treated with the Erg inhibitor, E4031 (10 µM), for 24 h with and without apoptosis inhibitor, Q-VD-OPh (20 µM) and viability was measured using an ATP detecting viability assay. Data presented as mean ± SEM (N=3), one-way ANOVA, Tukey post-hoc test.</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

The circadian clock transcriptional network is synchronized with the cell cycle.

<p>(A) Schematic illustrating the feedback loops of the circadian clock oscillators. (B) Plot of the θ-value for core circadian genes in HeLa-Fucci cells (p-value≤0.001). (C) Protein expression levels of core components of the circadian clock in HeLa-Fucci cells analysed by correlating fluorescent immunostaining intensity to cell cycle phase determined by Fucci reporters or DNA content (DAPI); bars represent the mean of the logarithmic intensity of cells relative the average mean logarithmic intensity of all cells. Error bars denote SEM. (D) Comparison between cell cycle oscillating transcripts in HeLa-Fucci cells (FDR≤0.001) and a published circadian clock transcriptome in non-proliferating liver cells[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188772#pone.0188772.ref055" target="_blank">55</a>]. (E) Plot of the θ-values for core circadian genes in HeLa-Fucci cells (FDR≤0.001) versus the circadian peak values in liver cells reported by Yoshitane H, <i>et al</i>. 2014. (F) Proposed model of the integration of cell cycle, circadian clock and genes associated with development found to be synchronized with the cell cycle.</p

Inhibition of Erg results in cell volume increase and rupture.

<p>(A) Images from time-lapse movies of mESCs treated with Erg inhibitor, E4031 (10 µM). (B) Mass redistribution measurements of mESCs treated with different concentrations of clofilium. (C, D) Time-lapse images of fluorescent reporter for actin (Lifeact-mCherry) (C) and myosin MHCII (MHCIIA-GFP) (D) in a bleb. Kymographs (far right) of a bleb showing actin and myosin intensity over time. (E, F) Time-lapse images of actin (Lifeact-mCherry) during Erg inhibition by fluorescent microscopy (E) and TIRF microscopy (F). Scale bars in C and D are 4 µm and 10 µm in E and F. Time bars in the kymographs (C/D, vertical) are 100 s. Time stamps in C/D are in seconds and in E/F in hours.</p