Supplemental Material

This article contains the following supporting material:

• Figure 1 - Constitutively active L61Rac1 mutant fails to rescue a number of cellular phenotypes of Cdc42^-/- MEFs. WT or Cdc42^flox/flox MEFs were transduced with retrovirus expressing EGFP or L61Rac1 mutant together with EGFP. The EGFP positive cells were isolated by FACS, and subjected to adeno-Cre treatment. (A) L61Rac1 expression did not alter morphology or actin structure of Cdc42^-/- cells and did not change the cellular response to bradykinin or PDGF stimulation to form filopodia or membrane ruffles. In addition, L61Rac1 expression was insufficient to rescue the adhesion (B) or migration (C) defect of Cdc42^-/- cells despite the ability to promote adhesion (B) and migration (C) in WT cells. *p<0.05 between the indicated data pairs.

• Figure 2 - Effect of Cdc42 loss or gain of activity on cell spreading kinetics on plastic culture dishes. The cells were detached and plated 5 X10⁴ per 60mm tissue culture plate (Linbro/Titertek, Flow Lab. Inc.) at 37°C. Cell spreading was monitored continuously under phase contrast microscope (Leica DM IRB) equipped with an ORCA-ER C4742-95 camera (Hamamatsu). The assays were performed in triplicate.

• Figure 3 - Effect of loss of Cdc42 activity on wound healing migration. Cells were plated on the 60-mm-diameter dishes to confluence and grown in the presence of 10% FBS. Wound was introduced by scraping with a 20–200-μl pipette tip across the dish and photos were taken at the different time points after the introduction of the wound. The arrowheads indicate the cell edges during the time course.