Figure 6.

E-cadherin shedding is mediated by increased MMP-9 activity. (A) The indicated cells were treated overnight with PD 98059 (50 μM) or vehicle (control). Total RNA was isolated and expression of MMP-9 mRNA normalized to 18S determined by quantitative RT-PCR. Data are representative of three independent experiments. (B) Cell were treated with PD 98059 or vehicle, and cell lysates were prepared in MAPK lysis buffer. MMP-9 activity was detected by zymography as described in Materials and Methods. Separate lysates were harvested and pretreated with MMP-9 inhibitor before being run on zymography gels. (C) Lysates were isolated from 3D cultures of vector control and K-Ras cells. Extracts were analyzed for MMP-9 activity as in A. (D) The indicated cells were treated with either 50 μM PD98059 or 20 μM MMP-9 inhibitor overnight. Lysates were immunoblotted with the C36 anti-E-cadherin antibody. Both inhibitors significantly reduced E-cadherin cleavage. (E) Confluent dishes of vector or K-Ras cells were treated overnight with the MMP-9 inhibitor or vehicle and TER measured as in Figure 5. MMP-9 Inhibitor increased TER only in K-Ras cells.