Fig. 3. Rac-mediates resistance to apoptosis and changes in tissue polarity through distinct mechanisms. (A) (top) Phase-contrast and confocal immunofluorescence images of colonies of control mammary acini (left panel; control), acini expressing EGFP-tagged N17Rac (middle panel; N17Rac) and acini treated with the Rac1 inhibitor NSC23766 (right panel; NSC23766) stained for 4 integrin, laminin-332, collagen IV and scribble (red). The data show that expression of N17Rac affects neither tissue integrity (compare regions highlighted by arrows in control phase-contrast images of acini with regions highlighted by arrows in images of N17Rac-expressing acini; top) nor tissue polarity (lower images; evidenced by similar basally localized 4 integrin; deposition of laminin-332 and collagen IV and intact cell-cell-localized scribble; compare images of control acini with images of acini expressing N17Rac). However, treatment of acini with the Rac inhibitor NSC23766 disrupted the integrity of acini (compare regions of image highlighted by arrows in phase-contrast images of control with images of NSC23766-treated acini) and severely perturbed tissue polarity, as evidenced by disturbed localization of 4 integrin, laminin-332, collagen IV and scribble (compare images of control with images of NSC23766-treated acini). Bar, 50 µm. (B) Representative immunoblot of GTP-Rac, Rac and E-cadherin in vector control MECs grown as 3D acini in comparison to MECs expressing EGFP-tagged N17 Rac and control acini treated with the specific Rac inhibitor NSC23766. The data illustrate that while N17Rac partially reduces GTP-Rac levels in 3D mammary acini, treatment with the Rac inhibitor decreases Rac activity to barely detectable levels. (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) Bar graphs illustrating increased percentage of apoptotic cells induced by treatment with Trail (1 µg/ml) (left) or Taxol (40 µM) right in 3D rBM differentiated MECs with significantly reduced Rac activity mediated by treatment with the specific Rac inhibitor NSC23766. The percentage apoptosis was calculated by scoring the number of activated caspase-3-positive MECs divided by the total number of MECs 24 hours following treatment with Trail or Taxol. Results are the mean ± s.e.m. of three to five separate experiments. ^*P0.05; ^**P0.01.