FIG. 1.

Epoxomicin induces protein ubiquitination and cell death associated with cytochrome c release, caspase activation, and nuclear apoptotic morphology in neocortical neurons. (A) Cortical neurons were treated with epoxomicin (Epoxo; 50 nM) or the control (Con) (dimethyl sulfoxide [DMSO], 0.1%) for the indicated periods. Western blotting was performed using an antibody recognizing mono- and polyubiquitinated proteins. Probing for β-actin served as a loading control. (B) Bright-phase, PI-positive neurons and the merged image in control and neurons treated with epoxomicin for 24 h. Scale bar, 50 μm. (C) Hoechst-stained neurons illustrating nuclear condensation and fragmentation in DMSO- and epoxomicin-treated samples. Scale bar, 20 μm. (D) Quantification of PI-positive neurons and caspase-3-like (DEVDase) activity following epoxomicin treatment. PI-positive nuclei were expressed as a percentage of total cells per field. A minimum of 300 neurons in at least three different fields were captured per well, and at least three wells were analyzed per time point (*, P < 0.05 compared to control). Caspase 3-like activity was assessed by measuring the cleavage of the fluorogenic substrate Ac-DEVD-AMC (10 μM). DEVDase activity was expressed as fold increase over the control. The data are the results from three measurements per well and three wells per time point (#, P < 0.05 compared to control). (E) Immunocytochemistry of cytochrome c (Cyt-c) in neurons. The redistribution of cytochrome c is observed in treated samples (scale bar, 10 μm). All data are means ± SEM from three wells; experiments were repeated three times from independent cultures with similar results.