TI - Sema4D -induced migration is mediated by Met . AB - To evaluate whether Met is functionally required for Sema4D -induced motogenic effect , we performed three-dimensional matrix assays by coculturing for 72-h aggregates of GN11 cells together with aggregates of Sema4D-secreting or mock-transfected COS cells in the presence or absence of the specific Met kinase inhibitor PHA-665752 ( PHA ; Fig 7 b ) . This highly specific small-molecule inhibitor has an IC50 for Met in the nanomolar range , >1,000 times higher than that for other receptor tyrosine kinases . Treatment of GN11 cells with 200 nM and 500 nM PHA , respectively , curbed the PHOSphorylation of Met down to levels comparable to cntr conditions ( Fig 7 a ) . In coculture experiments , many more GN11 cells migrated out of the aggregate in the presence of a source of Sema4D , as compared with mock treatment ( Fig 7 , b and c ) . Moreover , asymmetrical directional migration of GN11 cells pointed to a chemoattractive activity of Sema4D ( Fig 7 , b and c ) . Such an attractive effect was prevented when the Met inhibitor PHA was applied to the culture medium ( Fig 7 , b and c ) . These results were further confirmed by blocking Met activation with RNA interference technology . GN11 cells were infected with a lentiviral construct expressing both a Met-specific short hairpin RNA ( shRNA ) sequence and the marker GFP , under control of a separate promoter . As a specificity control , we used a construct expressing a mismatched Met shRNA sequence ( shRNA cntr ) . Infection efficiency was confirmed by visualization of GFP expression under a fluorescent microscope ( Fig 8 , a and b ) , and Met silencing was verified by Western blot analysis ( Fig 8 c ) . As shown in Fig. 8 c , infection with Met shRNA impaired the expression of Met in these cells , whereas cntr shRNA did not decrease Met expression compared with GN11 WT cells ( WT ) . PlexinB1 expression was not affected after infection ( Fig 8 c ) . To determine the biological significance of Met in Sema4D -mediated GN11 cell migration , we performed migration assays using the Boyden's chamber . As expected , cntr cells migrated toward a source of HGF as well as of Sema4D ( Fig 8 d ) . In contrast , Met-silenced cells displayed impaired ability in responding to either of the two chemoattractive factors ( Fig 8 d ) . These results show that Sema4D -induced motogenic activity of GnRH-1 cells is mediated by PlexinB1-Met complex .