Fig. 8. ARAP3 does not affect cell adhesion but impairs cell migration. The indicated Flp-In^TM T-Rex^TM HEK293 cell lines were treated with 2 µg/ml doxycycline for 16 hours to induce ARAP3 proteins, harvested and maintained in suspension in serum-free medium for 1 hour, then plated in quadruplicate in microwells (5000 cells/well) that were pre-coated with 2.5, 5, 10 or 20 µg/ml fibronectin. Cells were allowed to adhere for 35 minutes, wells were washed four times, and the relative numbers of adhering cells determined using an MTT assay (Materials and Methods). Data are the mean percentage ± s.d. of maximal levels of adhesion (20 µg/ml fibronectin) in two independent experiments. (B) Equivalent numbers of ARAP3 and R938L Flp-In^TM 293 cell lines that had been treated (+) or not treated (-) with doxycycline (Dox) and serum-starved for 16 hours were seeded (5x10⁴ cells in triplicate) on Transwell 8 µ pore filters (coated with 10 µg/ml fibronectin). Filters were suspended in serum-free medium and incubated at 37°C for 8 hours. Cells were removed from the upper side of filters and the relative numbers of cells migrating to the underside was determined by fixing and staining the cells with 1% crystal violet, extracting the dye and determining its absorbance at 590 nm. Data are means ± s.d. and are representative of three independent experiments. Statistical significance was assessed using an unpaired t-test; ^*P<0.05.