Figure 8.

MEK inhibitor UO126 prevents HGF-induced epithelial disruption in MDCK cells expressing deregulated ErbB2/Neu. Stable lines of MDCK cells were grown in collagen I. Five days later, HGF (5 U/ml) and UO126 (5 and 20 μM) were added either simultaneously as in A, or at a 2-d interval where pretreatment was required, as in B. Seven days later, cultures were fixed in 4% paraformaldehyde. Representative pictures were taken using bright field (2.5×) and phase contrast (10×) objectives. Live/dead staining was performed on cultures after treatment with UO126. A representative picture is shown as an insert. (C) Representative collagen cultures from B stained for E-cadherin and ZO-1 show restored tubular structures with E-cadherin and ZO-1 staining at cell-cell junctions. Confocal LSM510 (magnification, 63×). (D) HGF and NeuNT synergize to enhance Erk phosphorylation. MDCK cells expressing deregulated ErbB2/Neu (NT9) were serum starved for 18 h, and then stimulated with HGF (10 U/ml) for the indicated times. Cells were pretreated with the MEK inhibitor UO126 (10 μM) 1 h before HGF stimulation where indicated. Proteins from total cell lysate (50 μg) were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted for phospho-Erk and total Erk.