Fig. 7. ARAP3 inhibits spreading of HEK293 cells on fibronectin in a Rho GAP-dependent manner. (A) The rate of spreading of serum starved, doxycycline-treated (2 µg/ml for 16 hours) Flp-InTM 293 control cells (filled boxes) versus those expressing ARAP3 (filled triangles), R93 8L (filled diamonds), R527K (open diamonds), R527K/R938L (open boxes) and Y1399/1404F (open triangles) was evaluated by determining the percentage of cells elaborating lamellipodia-like protrusion(s) at the indicated times (minutes). >200 cells were scored at each time point. Data are the means ± s.d. of duplicate determinations from two experiments. (B) The indicated Flp-InTM 293 inducible cell lines were treated with doxycycline (2 µg/ml) for 16 hours, trypsinised, washed and suspensed in serum-free medium for 1 hour. Cells were plated on coverslips pre-treated with 10 µg/ml fibronectin and cells fixed and stained with crystal violet at the indicated times. Cell morphology was visualized by differential interference contrast (DIC) light microscopy. Representative images from two experiments are shown.