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Fig. 2. Rac activity is necessary for resistance to apoptosis in 3D mammary acini. (A) FACS analysis showing increased EGFP expression in MECs expressing the EGFP-tagged N17 Rac (P4) in comparison with vector control MECs (P3). (B) Representative immunoblot of GTP-Rac, Rac and E-cadherin in vector control MECs grown as 2D monolayers in comparison with MECs expressing EGFP-tagged N17 Rac. The data illustrate that N17Rac significantly reduces GTP-Rac levels in MECs. (C) Average relative specific activity of Rac in MECs calculated by densitometric analysis of immunoblots of GTP-Rac divided by total cellular Rac following E-cadherin normalization of data illustrated in B. (D) Representative immunoblot of phospho-Pak1 and total Pak1 in 2D monolayer cultures of control MECs and MECs expressing EGFP-tagged N17Rac demonstrating how loss of Rac activity also reduces Pak1 activity. (E) Bar graph depicting the average degree of reduction of Pak1 activity in MECs expressing EGFP-N17Rac in comparison with control MECs. (F) Dose-response curves of the percentage apoptosis, as determined by calculating the number of activated caspase-3-positive cells divided by the total cell number, showing how 3D rBM polarized mammary acini with reduced Rac activity are now more sensitive to both chemotherapeutic (taxol) and receptor-mediated (Trail) apoptotic stimuli. Results are the mean±s.e.m. of three to five separate experiments. *P
0.05 (C,E,F); **P0.01 (F).
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