FIG. 9.

hT-RPE-MycER/V12Ras cells rely on uPA's proteolytic activity for invasiveness. (A) uPA silencing reduces the ability of hT-RPE-MycER/V12Ras cells to invade Matrigel. hT-RPE-MycER/V12Ras cells were transfected with control or uPA-targeting siRNAs. After 48 h of 4-OHT treatment, hT-RPE-MycER/V12Ras cells (10⁵/sample) were incubated in the upper compartments of Boyden chambers with an additional layer of Matrigel (50 μg/ml) over collagen-coated 8-μm filters. After incubation for 3 h at 37°C, the ability to invade toward EGF (50 ng/ml) was measured by counting the cells on the lower-chamber side of the filter. (B) Only proteolytically active uPA can restore the ability of hT-RPE-MycER/V12Ras cells to invade Matrigel after c-Myc activation. As described above, hT-RPE-MycER/V12Ras cells were treated or not treated with 4-OHT and assayed in Boyden chambers with a Matrigel layer. Where indicated, cells were preincubated for 2 h with either full-length uPA or an uPA variant with the catalytic domain deleted (uPA1-158). Significance was calculated using the Student t test (** indicates a P value of <0.001).