Figure 6

PRL-3 promotes invasion by enhancing MMP2 activity. (A) MMP activity was required for PRL-3-mediated invasion. Cells were treated with 25 μM of GM6001 or DMSO (control) for 2 h, and analyzed for their invasion abilities with Matrigel-coated transwell chambers. The invasion assay was performed for three times independently. Top: Representative illustrations of LoVo cells invading to the underside of filters. Bottom: Total number of invasive cells. The experiments were repeated three times independently. Error bars represent standard errors of the mean value (*, P < 0.05). (B) PRL-3 increased gelatin hydrolytic activity of MMP2 in LoVo cells. Equal amount of conditioned serum-free media of LoVo-C and LoVo-P cells was subjected to a zymographic assay as described in Experimental Procedures. A small aliquot of calf serum containing MMP9 and MMP2 was included as the positive control. Bright bands contrasting to dark background represented the hydrolysis area of gelatin catalyzed by the MMP at the same molecular weight. (C) PRL-3 affected MMP2 and TIMP2 expression at both mRNA and protein levels. Equal amount of cell lysates (50 μg of protein) and RNA samples were subjected to Western blot and RT-PCR assays to analyze the expression of MMP2 and TIMP2, respectively.