FIG. 5.

Ectopic expression of NGB reduces cell mobility, attachment, and aggregation. (A) Reduced cell mobility was observed in cells overexpressing NGB. An MICS assay was used to directly measure cell migration. The migration is normalized to values obtained with pcDNA3 vector controls and is expressed in μM/h. Standard deviations for each cell line are shown. (B) NGB inhibits cell adhesion. NGB-59 and pcDNA3-rat schwannoma cells were incubated in the presence or absence of 100 μM zinc chloride overnight and then harvested by trypsinization. Equal numbers of cells were added to laminin-, fibronectin-, collagen IV-, and collagen I-coated 96-well plates. After 1 h, the number of adherent cells was determined using crystal violet staining and a spectrophotometric assay. NF2-transfected cells (NF2-19) were used as controls. The experiment was repeated three times. OD450, optical density at 450 nm. (C) JS1 cell aggregation was inhibited by NGB. Single-cell suspensions (500 μl; 5 × 10⁴ cells/ml in DMEM containing 0.5% BSA) were plated into each well of a 24-well low-binding plate. The plate was incubated at 4°C or 37°C on a rotating platform for 30 min. Photographs were taken using a Nikon N6006 camera (magnification, ×100).