Fig. 4. Activity of Rac1 and Cdc42 is not required for the formation of blebbistatin-induced protrusions. (A) Caco-2 cysts were treated for 12 hours with either blebbistatin alone or in a combination with the Rac inhibitor NSC 23766 (100 µM) or the Cdc42 inhibitor secramine A (20 µM). Pharmacological inhibition of Rac and Cdc42 did not prevent the development of blebbistatin-induced spikes. (B) Caco-2 cells growing on Matrigel-coated coverslips were infected with adenoviruses bearing either control EGFP, or EGFP-tagged dominant-negative N17Rac1 or N17Cdc42 and treated for 12 hours with blebbistatin (100 µM). Cells infected either with control virus or with dominant-negative Rac1 or Cdc42 mutants developed peripheral protrusions upon inhibition of myosin II (arrows). (C) Caco-2 cells were treated for 12 hours with either blebbistatin (100 µM) or vehicle, and activation statuses of Rac1 and Cdc42 were determined in cell lysates using pull-down assays. No increase in the levels of activated Rac1 or Cdc42 were observed in blebbistatin-treated cells.