Fig. 5. Rac-dependent Pak1 activity is necessary and sufficient for the resistance to apoptosis of mammary acini. (A) Representative immunoblots of phospho-Pak1, total Pak1, Pak-3, phospho-Pak4, total Pak4 and E-cadherin in rBM-ligated MECs plated as 2D monolayers or 3D mammary acini. The data indicate that Pak1 activity is significantly higher, although the abundance of Pak4 is higher in 3D the specific activity of Pak4 is substantially lower and Pak3 is non-detectable in 3D mammary acini in comparison with 2D monolayers of MECs. (B) Quantification of averaged experimental data shown in A of Pak1 specific activity, calculated by densitometric analysis of immunoblots of phospho-Pak1 divided by total Pak1 after normalization to E-cadherin. Similar results were obtained for HMT-3522 S1 and MCF10A nonmalignant MECs. (C) Representative immunoblots of phospho-Pak1 and total Pak1 in 3D mammary acini expressing the tail-less 4 integrin (4cyto) in comparison with control acini. The data demonstrate that Pak1 activity, but not expression, is regulated by (6)4 integrin signaling. (D) Quantification of averaged experimental data shown in C of Pak1 specific activity calculated as described above in B. (E) Dose-response curves illustrating the percentage apoptosis induced in 3D mammary acini following 24 hours of treatment with increasing concentrations of Trail (left) and taxol (right), calculated by scoring the number of caspase-3-positive cells divided by the total number of cells. Mammary acini with reduced Rac activity were sensitized to Trail and taxol-induced apoptosis (N17Rac vector) but their death-resistance phenotype was restored following ectopic expression of V12Rac. (F) Bar graphs illustrating how expression of V12Rac restores resistance to apoptosis induced by Trail treatment in 3D mammary acini expressing N17Rac, whereas expression of V12Rac H40, which cannot activate Pak, does not restore resistance. The percentage apoptosis was calculated by scoring the number of activated caspase-3-positive cells divided by the total number of cells. (G) Bar graph demonstrating that inhibiting Pak activity, by expressing PID significantly sensitizes mammary acini to Trail-induced death, analogous to that mediated by N17Rac. (H) Bar graph showing how expression of wild-type Pak1 can restore resistance to Trail treatment to 3D mammary acini expressing N17Rac. Results are the mean ± s.e.m. of three to five separate experiments. ^*P0.05; ^**P0.01.