Figure 2

SerpinE2 silencing in caMEK-expressing IECs decreases foci formation, growth in soft agar and migration. A- caMEK-expressing IECs were stably infected with lentiviruses encoding for a control shRNA (scrambled sequence) or encoding a serpinE2-specific shRNA. These stable cell populations were thereafter lysed at confluence and equal amounts of concentrated culture medium were analyzed by Western blotting with specific antibodies against serpinE2. The graph illustrates densitometric analysis performed with the western blot data shown on the left to determine the % of serpinE2 downregulation. The level of serpinE2 observed in shScrambled cells was set at 100%. A representative experiment of three independent experiments is shown. B- wtMEK/IEC-6 or caMEK/IEC-6 cells expressing either shScrambled or shSerpinE2 were seeded in a 6-well plate at 100 000 cells per well. Cells were harvested and counted. Values are means of 4 experiments ± SE. C and D- caMEK-transformed cells expressing or not shSerpinE2 were seeded on parental IEC-6 cell monolayer during 15 days. Thereafter, the cells were stained with crystal violet and images of colonies were acquired under light microscopy. The size of the foci was calculated using Image J software and expressed as % of shScrambled cells (control) which was set at 100%. E- Cells were cultured in soft agarose for 3 weeks before MTT. The number of colonies was determined using Image J software and expressed as % of shSrcambled cells (control) set at 100%. F- Cell migration to the undersurface of the polycarbonate membrane of Boyden chambers coated with fibronectin (FN) or vitronectin (VN) was evaluated 24 h after seeding, in presence of 20 mM hydroxyurea. Values were expressed as a % of shScrambled cells migrating on fibronectin. *, significantly different from shScrambled cells at p < 0.05 (Student's t test).