Fig. 3

RI-3 prevents mesothelial cell adhesion and invasion by SKOV-3 cells. HPMCs were purified from human omental specimens and cultured as described in the Methods. a-b. Representative images of HPMCs visualized by phase contrast microscopy (a) or stained for cytokeratin 8/18 (green) and vimentin (red) (b). Nuclei were stained blue with DAPI. Original magnification: 400x (a), 1000 x (b). c. HPMCs were seeded in 24-well plates and allowed to attach and form a monolayer for about 20 h prior to seeding GFP-SKOV-3 cells suspended in complete medium plus diluents (none), or 10 nM RI-3 at 37 °C, 5% CO₂. At the indicated times, cell associated fluorescence was assessed by a fluorescence plate reader. Data represent means ± SD of three independent experiments performed in triplicate with *P < 0.0001 vs none. d-e. Mesothelium invasion by SKOV-3 cells. HPMCs (5 × 10³ cells/well) were seeded in E-16-well plates in growth medium and allow to adhere for 20 h until they form a confluent monolayer (plateau curves). Then, SKOV-3 cells (2 × 10⁴ cells/well) suspended in growth medium plus/minus 10 nM RI-3 were seeded on mesothelial cell monolayer. The invasion of mesothelium by SKOV-3 cells was monitored in real-time as changes in Cell Index due to breaking of the monolayer integrity (d). Slopes represent the change rate of Cell Indexes generated in a 20–24 h time frame (e). Data represent mean ± SD from a quadruplicate experiment representative of 3 replicates