Expression of amino-terminal ezrin inhibits motility and invasion of carcinoma cells. (a) Transfected AC2M2 clones (see Fig. 1) were grown to confluence in 12-well NUNC tissue culture plates with 2 ml of 10% foetal bovine serum (FBS)/Dulbecco's modified Eagle medium (DMEM). Cultures were wounded by streaking cell monolayers with a 20 μl Eppendorf micropipette tip and monitored up to 24 hours, as described in the text. Representative fields photographed after 18 hours are shown. Vertical bars indicate the wound distance. Bar, 50 μm. (b) Transfected AC2M2 cells were subjected to an invasion assay, as described in the text. Cells (5 × 104) were over-layered in 200 μl of 0.5% FBS/DMEM medium on the transwell membranes (8 μm pore size), with 0.5 ml of complete medium in the lower chamber. After 36–48 hours, cells were fixed and stained with modified haematoxylin, and cells invading through the membrane were counted using ImagePro software, as described in the text. The numbers of invading cells were normalized to empty pCB6 vector group in each experiment. The results from at least three independent experiments were pooled, and expressed as the mean relative cell invasion ± standard deviation. Asterisks indicate (**) a significant increase or (*) a significant reduction in cell invasion compared with vector control, using a two-sided Fisher's t-test. P = 0.03 for WTC4**; P = 0.009 for NTC6*; and P = 0.02 for NTC7*.