Fig. 6. Pak permits activation of NF-B to mediate resistance to apoptosis in mammary acini. (A) Bar graph indicating how inhibiting activation of NF-B by treating mammary acini with SN50 peptide permits Trail-induced apoptosis. MECs were grown in rBM for ten days and treated with SN50 or inactive, scrambled SN50M peptide. Polarized acini were treated with Trail (1 µg/ml) and after 24 hours the acini were stained and quantified for activated caspase 3. (B) Bar graph illustrating how ectopically expressed wild-type Pak1 can restore resistance to apoptosis to N17Rac-expressing mammary acini treated with Trail (N17Rac), but the acini remain death sensitive if NF-B activation is prevented by pre-incubation with SN50. 3D mammary acini were infected with adenovirus, pre-incubated for 24 hours with either SN50 to inhibit NF-B activation or its non-active analogue SN50M, and treated with Trail (1 µg/ml) for 24 hours. The percentage apoptosis for (A) and (B) was calculated by scoring the number of activated caspase-3-positive cells divided by the total cell number. (C) Confocal immunofluorescence microscopy images showing NF-B p65 nuclear translocation in response to treatment with Trail (90 min) in 3D mammary acini expressing either vector (control), the Pak activity inhibitor (PID), N17Rac (N17Rac) or N17Rac and wild-type Pak1 (N17Rac/Pak1 WT). Note the presence of high nuclear levels of p65, as indicated by "n" and identified by arrow, in response to Trail stimulation in control and N17Rac/Pak1-WT-expressing mammary tissues and decreased levels in acini with reduced Rac or Pak activity. Bar, 10 µm. n, nucleus. (D) Quantification of nuclear p65 in 50-100 representative images as shown in C. (E) Bar graph showing how expression of a wild-type p65 transgene restores Trail-induced death-resistance to apoptosis-sensitized N17Rac- and PID-expressing mammary acini. (F) Bar graph showing how inhibiting activation of NF-B through expression of the IBM super-repressor permits Trail-dependent induction of death in 3D mammary acini despite elevated levels of Pak1. Results are the mean ± s.e.m. of three separate experiments. ^*P0.05; ^**P0.01; ^***P0.001.