Reduced <i>Mgat2</i> expression in NB cells enhances cell-cell adhesion.

<p>Representative DIC images acquired from 3 cell dissociation experiments of NB_1 (left panels), NB_1(-<i>Mgat2</i>) (middle panels), and NB_1(-/+<i>Mgat2</i>) (right panels) cell lines <i>(A)</i>. Cell clusters of more than 10 cells, as encircled in black, were examined. The bar graph represents the mean area of cell clusters for each cell line <i>(B)</i>. *<i>P</i><0.01.</p

Lack of <i>Mgat2</i> expression in NB cells reduces tumorigenic potential.

<p>Representative DIC images of cell colonies from NB_1 (left panels) and NB_1(-<i>Mgat2</i>) (right panels) cell lines obtained from the anchorage independent cell growth assay conducted on 3 separate occasions <i>(A)</i>. Mean values of cell colony area <i>(B)</i> from at least 98 colonies, and cell colony number <i>(C)</i> per experiment. *<i>P</i><0.01.</p

Cell growth and proliferation of NB cells were slowed by lowering the expression of <i>Mgat2</i>.

<p>The bar graph represents the number of cells for NB_1 and NB_1(-<i>Mgat2</i>) at 2, 3, and 4 days of growth from 4 experiments performed in triplicate (<i>A</i>). The inset, shows fold increase per day. *<i>P</i><0.02. The cell proliferation assay were conducted 3 times in triplicate for the various NB cell lines (<i>B</i>). *<i>P</i><0.01. Cytolysis was measured using the LDH assay for NB_1 and NB_1(-<i>Mgat2</i>) cells (<i>C</i>). No significant difference was observed at <i>P</i><0.05.</p

Characterization of Kv3.1 proteins expressed in B35 cells.

<p>Western blots of wild type (Wt), N220Q, N229Q, and N220Q/N229Q Kv3.1 proteins. Kv3.1 proteins were detected when heterologously expressed in B35 cells (A). Arrows and lines denote the type of <i>N</i>-glycan attached to the Kv3.1 protein. Assignments of the various glycosylated and unglycosylated Kv3.1 proteins were based on immunoband shifts produced by glycosidase treatment. N220Q and N229Q proteins were digested (+) and undigested (−) with neuraminidase (B), PNGase F (C) and Endo H (D). A solid line on image indicates that samples were run on a different blot (B). The numbers adjacent to the Western blots represent the Kaleidoscope markers (in KDa). Similar migration patterns were observed on at least three separate Western blots.</p

Aglycoform displayed slower activation kinetics of ionic currents than glycoform.

<p>Whole cell currents for glycosylated (A, middle panel; B, top panel) and unglycosylated (A and B, bottom panels) Kv3.1 proteins were elicited from the indicated voltage protocol (A, top panel). Whole cell currents were scaled for inactivating (A) and non-inactivating (B) current types from B35 cells expressing glycosylated and unglycosylated Kv3.1 proteins. Right panels show traces at expanded time scales and grey lines denote currents at +40 and +60 mV. Traces were scaled to show differences in activation kinetics. Conductance-voltage (g/gmax) curves of both inactivating (C) and non-inactivating (D) current types for glycosylated and unglycosylated Kv3.1 channels. Rise times of inactivating (E) and non-inactivating (F) currents types. <i>n</i> represents number of cells.</p

NB cells with <i>Mgat2</i> silenced are less sensitive to chemotherapy drugs.

<p>The percent of viable cells after treatment of cells with cisplatin and doxorubicin were determined from at least 3 experiments. *<i>P</i><0.02.</p

Silencing of <i>Mgat2</i> minimizes cell invasiveness of 3D spheroids derived from NB cells.

<p>Selected micrographs of spheroids after 13 days of invading into matrix from NB_1 and NB_1(-<i>Mgat2</i>) (<i>A</i>). Cell invasion was calculated by dividing invasive area by the sphere area (<i>B</i>). The invasive area is between the inner and outer black traces. The white dotted line overlaid on the inner black line encircles the sphere area. The bar graph represents the cell invasion of at least 130 spheroids from 3 experiments conducted with the NB cells (<i>C</i>). *<i>P</i><0.00001.</p

Cell morphology of NB cells are altered by silencing <i>Mgat2</i>.

<p>Adhered cell growth of the NB_1 and NB_1(-<i>Mgat2</i>) cell lines were acquired at different time periods, 18 and 26 h, as shown in the representative DIC images (<i>A</i>). Bar graphs show the length of outgrowths (upper panel), and number of outgrowths per cell (lower panel) at both time points (<i>B</i>). The experiment was conducted 3 times and at least 207 outgrowths and 93 cells were analyzed. *<i>P</i><0.01.</p

Spatial arrangement of glycosylated and unglycosylated forms of Kv proteins in B35 cells.

<p>Microscopy images were acquired in TIRF (left panel), DIC (middle panel), and wide-field (right panel) modes of EGFP tagged wild type Kv3.1b heterologously expressed in B35 cells (a). TIRF images (middle panels), along with their accompanied DIC images (outer panels), are shown for wild type Kv3.1a (b, upper row) and Kv1.1 (c, upper row) proteins, and also for N220Q/N229Q Kv3.1a (b, lower row) and N207Q Kv1.1 (c, lower row) proteins. Representative scale bar (5 μm) was identical for all images. White and gold arrows point to EGFP tagged Kv proteins in the outgrowth and cell body, respectively.</p

Ionic currents of the aglycoform inactivated to a lesser extent than glycoform.

<p>Whole cell currents were elicited from the shown voltage protocol (A, left panel) for B35 cells expressing glycosylated (A, right panel) and unglycosylated (B) Kv3.1 proteins. Traces were scaled to show differences in inactivation kinetics. Grey lines denote currents at +40 mV.</p