Figure 2

In vitro behavior of cathepsin L antisense clones. (A) Invasion assay of cathepsin L sense and antisense clones. Data represents the number of cells per filter (counted with a 40X objective) that had crossed a Matrigel barrier during 24 hours incubation at 37°C. Triplicate wells were run in triplicate. Analysis of variance (ANOVA) showed there was a significant difference between antisense clones (AS) and sense clones (S) (* P < 0.001). (B) Migration of cathepsin L sense and antisense clones in Boyden chamber. Data is as for invasion, but with no Matrigel barrier. Migration difference was significant by Student t test (*P < .05). (C) Radial migration with sedimentation chamber. Cells (2 × 10³ cells per well) were plated on glass slide wells in triplicate using a cell sedimentation chamber. Chemokinetic bFGF (Sigma, 25 ng/ml) was used in these experiments. Cell colony diameters were imaged from 10X objective microscopic views. Data represent the average differences in cell colony diameter (mm) following 24-hour incubation on laminin-coated wells. Experiments were conducted in triplicate, and a significant difference was found between control and antisense clones by Student t-test analysis (*P < 0.01). (D) Morphological appearance of typical clones with light microscopy. Cells were plated on glass coverslips overnight and then images were taken using a 10 X objective. A) Control PC2 cells B) Cathepsin L antisense (AS5) cells. (E) Adhesion of cathepsin L antisense clones to extracellular matrix proteins. Plastic 96-well plates were coated with extracellular matrix proteins. Fibronectin and laminin were used to pre-coat wells with 10 μg/ml. Collagen I was pre-coated at 100 μg/ml. Cells (2 × 10⁴ per well) were added prior to incubation for specified times. Wells were washed with PBS twice, cells fixed with 4% paraformaldehyde, and stained with crystal violet prior to reading absorbances (A₅₉₅) of Triton X-100 (1% in PBS) lysed cells. Triplicate wells were run in triplicate and averaged. No significant difference was noted by Student's t test.