Fig. 9. Ectopic Pax3 expression induces expression of endogenous c-met and HGF induces epithelial-mesenchymal phenotypic reversion of Pax3^flag-expressing cell aggregates. Saos-2 cells were infected with either Ad-ßgal or Ad-Pax3flag viruses and harvested at the time points indicated. (A) Expression of c-met and Pax3^flag transcripts were assessed by semi-quantitative RT-PCR, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the same samples as a control. (B) Expression of c-met and Pax3^flag proteins was determined by western blot analysis with antibodies specific for each protein. Actin levels served as a loading control. (C-F) Phase-contrast images of Saos-2 cells infected with Ad-Pax3^flag (C,D) and Ad-ßgal (E,F) adenovirus. At three days postinfection cells were exposed to conditioned media containing six scatter units of HGF/SF (D,F) or control conditioned media (C,E) and images of live cells were captured 24 hours later. Exposure of Ad-Pax3^flag-infected cells to HGF/SF led to a complete dissociation of cell aggregates with many cells exhibiting an elongated motile appearance (arrowheads in D). Bars, 80 µm.