Tiam1 and talin are required for adhesion-induced Rac1 activation and cell spreading. (A) U251 cells transfected with the indicated siRNAs were seeded onto FN under serum-free conditions. Rac1 activity was measured by pull-down with PAK-CRIB. Depletion of either talin or Tiam1 impaired transient Rac1 activation upon adhesion. (B) Plots of the quantified data from A. (C) Non-transfected U251 cells were stained at 30 min after seeding with anti-phosphotyrosine (pY) and anti-Tiam1 antibodies. Bars, 10 µm. (D) Images of Tiam1- or talin1-depleted cell spreading on FN. The cells were stained with anti-GFP antibody (green) and phalloidin (red). Bar, 10 µm. (E) Quantification of spreading area and perimeter length during spreading of U251 cells. Results are representative of at least four experiments. Data represent the means ± SD of three independent experiments. n > 100. *, P < 0.05; **, P < 0.01 versus control cells in each time point (B, Student’s t test; E, Tukey’s HSD).