Figure 7
Participation of EGFR in LPA-induced IL-8 production, cell proliferation, migration and invasion. A. Caov-3 cells cultured in 6-well plates were starved and stimulated for 16 hours with LPA (10 μM) in the presence of indicated concentrations of AG1478. Levels of IL-8 released to culture supernatants were analyzed by ELISA. B. Caov-3 and SKOV-3 cells were cultured in 6-well plates with serum-free medium supplemented with EGF (25 ng/ml) or with 5 μM LPA in the presence of AG1478 (1 μM) or vehicle for 1-3 days. The cell numbers from triplicate wells were determined daily with a coulter counter. C. Caov-3 cells were grown in 60 mm dishes to confluence and starved before scratches were made with sterile pipettes. The cells were incubated for 16 hours with BSA or LPA (5 μM) in the presence of AG1478 (1 μM) or vehicle. Microscopic images (76×) were captured at 0 and 16 hours after LPA exposure. D. The migration and invasion of SKOV-3 cells in response to EGF (25 ng/ml) or to LPA (1 μM) in the absence or presence of AG1478 (1 μM) were analyzed using transwells and transwells coated with growth factor-reduced basement membrane matrix, respectively. SKOV-3 cells were starved with or without AG1478 before trypsinization. The cells (2.5 × 104 cells/0.5 ml for migration and 1 × 105/0.5 ml for invasion) were loaded to the upper chambers and allowed to migrate for 6 hours or invade for 20 hours. The migrated and invaded cells on the underside of transwells were stained with crystal violet and counted under microscope. Results were average numbers of migrated or invaded cells/transwell from triplicates (mean+ SD). The data shown was representative of three independent experiments.