Fig. 3. Effects of RhoG, Dock180 and Rac knockdowns on cell spreading. (A) MEFs transiently transfected with pSuper knockdown vectors plus GFP (ratio 40:1) were suspended in serum-free medium for 30 minutes then replated on coverslips coated with 10 µg/ml fibronectin (FN). At the indicated times, cells were fixed and stained for F-actin. Upper panels: left, representative blot of RhoG and Rac knockdowns; right, Alexa-Fluor-568–phalloidin staining of GFP-positive cells replated on FN for 25 minutes. Scale bar: 10 µm. Lower panel: quantitation of cell spreading. Average spread area of GFP-positive cells from randomly selected fields was measured and normalized to the size of unspread cells. Values are means ± s.e.m.; n>30 cells from two separate experiments. (B) Left panel: quantification of cell area after RhoG, Dock180 or Rac1 knockdown at 15 minutes after replating; ^*P<0.01, ^**P<0.001 (Student's t-test). Right panel: representative blot of Dock180 knockdowns. (C) Left panel: HeLa cells were transiently transfected and replated on FN-coated coverslips as described above. Spread area of GFP-positive cells was quantified from F-actin staining. Values are means ± s.e.m.; n>30 cells from two separate experiments. Right panel: representative blot of RhoG knockdown in HeLa cells. (D) Cell-adhesion assay. MEFs transfected with pSuper or RhoG shRNA were replated for 10 minutes in wells coated with the indicated amounts of FN or poly-L-lysine (pLL). Bound cells were quantified as described in the Materials and Methods. Values are means ± s.d.; n=6 from two independent experiments.