Figure 3. Loss of Cdc42 in HRasV12 transformed cells results in reduced cell proliferation and anchorage-independent growth.

(a) Equal cell numbers were plated following cdc42 deletion and cells were counted by trypan blue exclusion every three days for 9 days. Curves represent at least two independent experiments performed in triplicate. (b) Cdc42-proficient and –deficient HRasV12 cells were grown in a 0.3% agarose solution. Experiments were performed in triplicate and 9 independent fields were counted for colonies. (c) Exponentially growing cells were pulsed with 5-bromodeoxyuridine (BrdU) for 1 hr. prior to harvest. Cells were stained with anti-BrdU and propidium iodide (PI) and analyzed by flow cytometry. Graphs represent at least three independent experiments. (d) Cell lysates were immunoblotted for the cell cycle regulators, Cyclin D1 and p16^ink4a. Gapdh served as a loading control.