Fig. 5

ATP repletion, colchicine-triggered apoptosis, and caspase activity in DG-treated CGC. (A) Medium of CGC was supplemented with glucose (10 mM) or not. Neurons were then treated with colchicine (1 µM). Deoxyglucose (DG, 2 mM) was added to all cultures 8 hr after colchicine treatment. ATP was measured 9.5 hr after addition of colchicine (1.5 hr after DG); caspase activity and apoptosis were quantitated after 18 hr exposure to colchicine (8.5 hr after DG). As a control, neurons were incubated with colchicine alone. Data from this incubation (see Figs. 1, 4) were used as 100% reference values. All data shown are expressed as a percentage of colchicine-alone values and are means from three different experiments. (B) CGC were incubated in the absence or presence of glucose and of different inhibitors as described above. After 18 hr, cell lysates were prepared and analyzed by Western blot for caspase-3 processing. Recombinant caspase-3 was used as standard. (C) CGC were exposed to colchicine for 18 hr either directly in the presence of DG, or in the presence of DG plus glucose. Phosphatidyl-serine (PS) translocation was stained in live neurons with annexin V and imaged by confocal microscopy. Plasma membranes of >90% of all neurons were impermeable to ethidiumhomodimer at that time point. Microtubules and chromatin structure were examined in parallel cultures after fixation and staining with anti-βIII-tubulin and H-33342.