Fig. 8

Reducing TGF-β signaling in oncogenic Ras transformed MDCK cells by SPSB1 suppresses cell migration and invasion. 21D1 cells (a and c) were co-transfected with eGFP construct and FLAG-SPSB1 or pEF-BOS for 48 h. Cell monolayers (a) were then scratched as described in materials and methods, treated with ± TGF-β (2 ng/ml) and phase contrast/fluorescence images were recorded at 0 and 24 h post-scratching. Total cell number (denominator) and eGFP expressing cell number (numerator) that migrated into the wounded area were counted and represented as relative cell migration in B. Similar results were obtained in three independent experiments. c Following DNA transfection, cell were collected and re-seeded into the upper chamber of 8 μM pore matrigel-coated transwell plates (matrigel 1:1 mixed with DMEM, 70 μl/well) ± TGF-β (2 ng/ml) as indicated for another 24 h. Cells that migrated to the bottom side of the upper chamber were then fixed and stained with Hoechst dye. Images were taken using a fluorescence microscope (magnification = 20×) in 4 random fields. Total cell number (denominator) and eGFP expressing cell number (numerator) that migrated to the bottom side of the upper chamber were counted and represented as relative cell migration. Bars represent the mean ± S.D. of triplicate wells from one of three representative experiments