Effects of Rac1 targeting on cell cycle and apoptosis may involve the PAK and Akt pathway. (A) BL41cells containing the p53ts mutant were treated with the Rac1 inhibitor, NSC23766, or transduced with a Rac1N17 mutant and immunoblotted for phospho-PAK1 and phospho-Akt (left panel), and potential effectors of the MAP kinase module (ie, ERK, JNK, p38; right panel) and their respective total protein levels. Both left and right panels were from the same immunoblot, and β-actin served as a loading control for both panels. Immunoblot bands were quantified by densitometry, and untreated controls were set to 1. (B) BL41 cells were transduced with lentivirus encoding either a YFP-tagged shRac1 construct or scrambled sh control (shSCR) in conjunction with retrovirus encoding either GFP-tagged caPAK, GFP-tagged caAkt, GFP-tagged Akt-KD, or GFP alone. Cells were harvested for flow cytometry 48 hours after infection to determine the rate of apoptosis in the YFP/GFP-copositive population by staining with Annexin V and 7AAD. (C) BL41 human B-lymphoma p53 mutant temperature-sensitive cells were incubated at either 32°C (p53 wild-type) or 37°C (p53 mutant) for 24 hours before a 3-hour pulse with 20μM wortmannin or 50μM LY294002. Cells were then harvested and subjected to GST-Pak pull-down assay and immunoblotted on the same day for Rac1, pAkt Ser473, total Akt, and Gapdh. The data are representative of 6 different experiments and error bars represent SD.