Figure 3. Cellular validation of G04 as a specific inhibitor of RhoA activity.

(A) NIH3T3 cells were treated with G04 at the indicated concentrations for 24 hours in serum-free media. Cells were subsequently stimulated with or without 10% calf serum for 15 min and were subjected to GST-Rhotekin or GST-PAK1 effector domain pull-down assays, and the activities of RhoA, Cdc42 and Rac1 were examined. Blotting of the respective total cell lysates was carried out in parallel. Relative amounts of GTP-bound form of the GTPases were quantified by densitometry measurements and normalized to those of the unstimulated cells. (B) Effect of G04 on cell stress fiber and focal complex assembly. NIH3T3 cells were treated with or without 30 μM G04 in serum-free media for 24 hours, and subsequently were stimulated with 10% calf serum for 15 minutes. Medium containing G04 was removed and cells were washed for 3 times. The cells were then fixed 6 and 24 hours after the wash, respectively, and stained with Rhodamine-phalloidin for F-actin and anti-vinculin for focal adhesion complexes. Images shown are representative of more than 100 cells examined. (C) G04 treatment affects signaling downstream of RhoA, but not that of Rac1/Cdc42. Western blots are of p-PAK and p-MLC and relevant controls of NIH 3T3 cells treated with G04 at indicated concentrations in serum-free media and subsequently stimulated by 10% calf serum for 10 min. (D) A comparison of G04 and ROCK inhibitor, Y-27632. The relevant control NIH3T3 cells or cells treated with G04 or Y-27632 of indicated concentrations were stimulated with 10% calf serum for 10 minutes prior to blotting for p-MLC.