Effects of perifosine on ROS generation ( A ) and GSH levels ( B and C ) and effects of DEM on DR4 and DR5 expression in the absence and presence of NAC ( D ). A, M4e cells were loaded with DCF-DA and then exposed to the indicated concentrations of perifosine (PRFS) or H2O2 (positive control). At the indicated times post addition of the agents, ROS generation was monitored as described in "Materials and Methods". B and C, M4e cells were treated with the indicated concentrations of perifosine or DEM (B) for 9 h or treated with 10 uM for the given times or 1 mM DEM for 9 h (C). The cells were then subjected to GSH assay as described in "Materials and Methods". D, M4e cells were treated with 1 mM DEM in the absence and presence of 5 mM NAC for the indicated times (NAC was added to the cells 30 min before addition of perifosine). The cells were then lysed for preparation of whole-cell protein lysates and subsequent detection of the indicated proteins using Western blot analysis.