FIG. 8.

Autophagy is not active at time points associated with epoxomicin-induced cell death. (A) WT and puma^−/− cortical neurons were treated with epoxomicin in the presence or absence of E64-D/pepstatin A (E/P; 10 μg/ml). Western blotting was performed for LC3 and p62. β-Actin was used as a loading control. (B) WT and puma^−/− neurons were transfected with GFP-LC3, and 24 h posttransfection, the neurons were treated with epoxomicin (50 nM) or bortezomib (100 nM) for 24 h or serum starved (Starv.) in Hanks' balanced salt solution (HBSS) for 4 h in the presence and absence of 1 mM autophagy inhibitor 3-methyl adenine (3-MA). Scale bar, 10 μm. (C) Quantification of the percentage of GFP-LC3 cells positive for puncta in neurons treated as described in panel B above (n = 200 to 300 cells/treatment). Similar results were observed in two independent experiments. (D and E) WT and puma^−/− cortical neurons were treated with epoxomicin (D) or bortezomib (E) in the presence or absence of 3-MA. Cells with condensed nuclei were expressed as a percentage of total neurons in a field. All data are means ± SEM. *, P < 0.05 compared to the WT control (Con) (ANOVA and Tukey's post hoc test). Experiments were repeated three times from independent cultures with similar results.