Morphological analysis of astrocytes cultured on glass with the additional of soluble factors.

<p>(A) Percentage of cells with protrusions. Control (N = 79), PDGF (N = 81), laminin (N = 68), bFGF (N = 71), LIF (N = 74). Immunofluorescence images of (B) control and (C) astrocytes treated with LIF stained for GFAP (green) and DAPI (blue).</p

Tortuosity of astrocytes.

<p>(A) Immunofluorescence image of an astrocyte on HBMEC-derived ECM. Endothelial cells were grown to confluence on glass-bottom dishes, and removed from the dish with a lysis buffer containing 0.5% Triton X-100 and 20 mM NH₄OH in PBS. (B) Immunofluorescence image of an astrocyte on 50 μm inner diameter fibronectin rings. Astrocytes tended to trace the rings and have smaller cell bodies like those seen in co-culture. Fibronectin (red), GFAP (green). (C) Tortuosity of astrocytes on surface coatings and in co-culture. The tortuosity (τ) is given by τ = <i>l</i>/c where <i>l</i> is the arc length of the processes and c is the shortest end-to-end distance. For a straight line τ = 1, whereas for a circle τ = ∞. While all of the surface coatings result in very small increases over the control, the astrocytes in co-culture have significantly higher tortuosities. Statistical significance was determined using a student’s t-test. ***P≤0.01, **P≤0.05, *P≤0.1.</p

Influence of soluble factors on astrocyte morphology.

<p>Data represent mean ± SE. Statistical significance was determined using a student’s t-test. ***P≤0.01, **P≤0.05, *P≤0.1. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092165#pone-0092165-g004" target="_blank">Figure 4</a> for the number of cells analyzed.</p

Influence of ECM coating on astrocyte morphology after 24 hours.

<p>Fluorescence images of astrocytes stained for GFAP (green) and DAPI (blue) on (A) glass, (B) collagen I, (C) collagen IV, (D) fibronectin, (E) matrigel, and (F) co-culture on a confluent monolayer of HBMECs. (G) Astrocyte (from panel (F)) seeded on a confluent monolayer of HBMECs, stained for GFAP (green), DAPI (blue), and ZO-1 (red). (H) The percentage of cells with protrusions. Total number of cells analyzed: uncoated (N = 103), collagen I (N = 85), collagen IV (N = 61), fibronectin (N = 63), matrigel (N = 54), co-culture (N = 58).</p

Influence of surface coatings on astrocyte morphology.

<p>(A) The cell area defined by the area of the cell body. (B) the cell diameter is overall size defined by the diameter of the smallest circle that can enclose the cell and all of its processes. (C) The protrusion length is the total length of all protrusions. (D) The degree of branching is the number of branch points divided by the number of primary protrusions. (E) The number of primary protrusions represents the number of protrusions emanating from the cell body. (F) The number of secondary protrusions represents protrusions emanating from primary protrusions. (G) The number of tertiary protrusions represents protrusions emanating from secondary protrusions. (H) The number of branch points represents the sum of secondary and other higher order protrusions (equivalent to the number of bifurcations). Data represent mean ± SE. Statistical significance was determined using a student’s t-test test. ***P≤0.01, **P≤0.05, *P≤0.1. Only cells with astrocyte-like morphology were analyzed (the total number of cells and the fraction of cells with astrocyte-like morphology are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092165#pone-0092165-g001" target="_blank">Figure 1</a>).</p

NKCC1 activity is necessary for GB cell invasion in vitro and its inhibition leads to formation of less invasive tumors in vivo.

<p>Quantification of transwell invasion assays of primary-cultured GB cells exposed to increasing doses of the NKCC1 inhibitor bumetanide (A) or transduced with NKCC1 shRNA (B); exposed to 10 µM of the KCC inhibitor DIOA (C) or stably transduced with KCC4 shRNA (D). Insets show schematic representation of the experimental design in (A) and (D). (E–F) Orhtotopic in vivo tumors formed by NKCC1shRNA cells were significantly larger and less invasive than control cells. Inset shows NKCC1 knockdown by protein expression. (G) Representative images of DAPI-stained coronal sections of mouse brains, after the implantation of control shRNA (left panel) or NKCC1 shRNA (right panel) cells. (G′) Confocal images of human-specific Nestin positive cells migrating across the corpus callosum at the area in the dotted square in (G). These results suggest that NKCC1 expression is necessary for efficient GB cell migration in vivo. Scale bars, 500 µm in low magnification panels and 20 µm in high confocal images panels. Bars represent mean ± SEM. * <i>p</i> value<0.05; ** <i>p</i><0.005.</p

EGF promotes phosphorylation of NKCC1, and activation of the PI3K-Akt pathway is necessary for EGF-mediated WNK3 phosphorylation.

<p>(A) Treatment of GB cell lines NS 318 and NS 567 with EGF (30 ng/ml) stimulates phosphorylation of NKCC1. Exposure of cells to EGF (30 ng/ml) for 10, 30, and 60 min show a time-dependent course of NKCC1 phosphorylation. Also, exposure of NS 567 to 60 ng/ml of EGF shows higher levels of phosphorylation than phosphorylation levels at the same time point at 30 ng/ml showing a dose-dependent effect. A line plot is presented with the quantification of the ratio of p-NKCC1/NKCC1. (B) Activation of PI3K is necessary for phosphorylation of NKCC1 after stimulation of HEK-293 cells with EGF. HEK-293 cells were serum starved overnight and were incubated with wortmannin (WM) for 30 min prior to stimulation with EGF for 30 min. Total cell lysate (150 µg) was immunoprecipitated with T4 antibody before immunoblotting with anti-phospho NKCC1 antibody (top panel) or T4 antibody (bottom panel). (C) Activation of PI3K is necessary for increased phosphorylation of WNK3 after stimulation with 30 ng/ml EGF. After overnight serum starvation, HEK-293 cells were incubated with WM for 30 min prior to stimulation with EGF for 30 min. Total cell lysate (150 µg) was immunoprecipitated with WNK3 antibody before immunoblotting with anti-phosphorylated Akt substrate (αPAS) antibody (top panel) or WNK3 (bottom panels). (D) Total cell lysate samples (25 µg) were also resolved by SDS-PAGE and blotted with phospho-Akt (threonine 473, top panel) and Akt (bottom panel) antibodies to show inhibition of Akt phosphorylation after PI3K inhibition. A line plot is presented with the quantification of the ratio of p-NKCC1/NKCC1, αPAS/WNK3, and p-Akt/Akt. (E) Akt phosphorylation motif, top panel. Middle panel shows the alignment of the sequences of rat and human WNK3 showing conservation of the Akt phosphorylation motif. Bottom panel shows sequence of WNK1, which is phosphorylated by Akt (bottom panel) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001320#pbio.1001320-Jiang1" target="_blank">[29]</a>. Conserved residues are highlighted in red letters.</p

NKCC1-Ezrin association affects net contractile moments and projected cell area.

<p>(A) Immunoprecipitation of NKCC1 in two GB cell lines shows that Ezrin and actin are associated with NKCC1. (B) Immunoprecipitation of Ezrin pulls down NKCC1 in several GB cell lines. (C) Cells that overexpress Ezrin-binding null NKCC1 generate lower contractile moments and have lower surface area. Black bars represent the transfected cells, and white bars represent the untransfected cells. Measured cells in the WT group (<i>n</i> = 16 untransfected cells; <i>n</i> = 11 transfected cells), and measured cells in the mutant group (<i>n</i> = 17 untransfected cells; <i>n</i> = 12 transfected cells). Bars represent mean ± S.E.M. * <i>p</i> value<0.001.</p

NKCC1 is highly expressed in GB tissue samples and primary human GB cells.

<p>(A) Quantification of NKCC1 immunoreactivity in a tissue microarray (TMA) containing samples of multiple glial tumors of different grades. The quantification was done using FRIDA software <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001320#pbio.1001320-Halushka1" target="_blank">[100]</a>. Red lines represent mean immunoreactivity levels. (B) Representative images of NKCC1 immunohistochemistry in tissue cores from the TMA including glial tumors of different grades, normal brain, and epithelial tissues, which express NKCC1 in the apical surface of epithelial cells as a positive control. (C) Immunoblot showing NKCC1 expression in multiple glioma cell lines. Information on the number of samples, age, and gender of the patient of origin of each tumor type can be found in Tables S1 and S2. (D) KCC4 expression by real-time PCR in different glioma cell lines. * <i>p</i> value<0.001.</p

NKCC1 knockdown decreases the net contractile moment and projected area in primary human GB cell lines.

<p>(A and B) Representative traction maps of GB cells stably expressing control shRNA or NKCC1 shRNA, respectively. The white line shows the cell boundary. Colors show the magnitude of the tractions in Pascal (Pa). Arrows show the direction and relative magnitude of the tractions. Scale bars represent 50 µm. Inset, phase contrast images of the respective cells on the elastic gel. Computed net contractile moment of GB cells expressing control shRNA or NKCC1 shRNA in (C), NS 561 (control shRNA <i>n</i> = 15 cells, NKCC1 shRNA <i>n</i> = 14 cells, <i>p</i> = 0.024) and (D) NS 501 (control shRNA <i>n</i> = 13 cells, NKCC1 shRNA <i>n</i> = 12 cells, <i>p</i> = 0.005). Net contractile moment is expressed in pico-Newton meter (pNm). Measurement of the projected cell area in µm² of (E) NS 561 (control shRNA versus NKCC1 shRNA, <i>p</i> = 0.01) and (F) NS 501 (control shRNA versus NKCC1 shRNA, <i>p</i> = 0.001). Data are presented as geometric mean ± SEM in log transformation.</p