Fig. 5.

ToxB and N17Rac1 each promote activation of caspases-9 and -3 in CGNs. (a) CGNs were incubated for 24 h in complete medium (containing serum and 25 mm KCl) in the presence of either vehicle (Veh; 2 μg/mL BSA in PBS) or ToxB (40 ng/mL). After incubation, cell lysates were resolved by SDS-PAGE on 15% polyacrylamide gels and proteins were transferred to PVDF membranes. The blots were then probed with an antibody that specifically detects the cleaved (active) form of caspase-9. (b) CGNs were treated exactly as described in (a) and lysates were western blotted with an antibody that recognizes both the pro- and cleaved (active) forms of caspase-3. The blots shown in (a) and (b) are representative of results obtained in three separate experiments. (c) CGNs were incubated for 72 h after infection with adenovirus expressing GFP and N17Rac1 (at a final titer of 50 infectious particles per cell). Following infection, cells were fixed in paraformaldehyde, blocked and permeabilized in 5% BSA and 0.2% Triton X-100, and immunostained with an antibody that specifically detects the active (cleaved) form of caspase-9 [Casp-9 (a)] and a Cy3-conjugated secondary antibody. Nuclei were stained with DAPI. The merged image shows that GFP- and N17Rac1-positive cells (indicated by the arrows) had condensed or fragmented nuclear morphology and displayed significant immunoreactivity for active caspase-9, whereas surrounding uninfected cells were largely healthy and devoid of active caspase-9. (d) CGNs were infected as described in (c) and were then immunostained for active (cleaved) caspase-3 [Casp-3 (a)]. As described above, most of the GFP- and N17Rac1-positive cells (indicated by the arrows) showed marked chromatin condensation and fragmentation and stained positively for active caspase-3. The images shown in (c) and (d) are composites of three to four different fields of CGNs infected with adenoviral GFP/N17Rac1. Ad-GFP control data not shown. Scale bars, 10 μm.