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

(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

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

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

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