IMMU-114 induces changes in mitochondrial membrane potential, generation of ROS, and release of AIF. (A) At the indicated time points (top panel), changes in mitochondrial membrane potential were determined by shift in the emission spectrum toward the left on staining with the dye, TMRE. At the indicated points (bottom panel), generation of ROS was measured by staining with dihydroethidium dye. Dihydroethidium becomes oxidized in the presence of ROS and intercalates within double-stranded DNA and fluoresces. Increased fluorescence measured by the shift to the right indicates increased production of ROS. (B) ROS scavenging by NAC inhibits IMMU-114–induced mitochondrial membrane depolarization and generation of ROS. Cells were pretreated with NAC (5mM) for 2 hours, followed by treatment with IMMU-114 overnight. (C) IMMU-114 induces phosphorylation of pERK1/2, JNK, and release of AIF in a time-dependent manner. Raji cells were incubated with IMMU-114 for the indicated times, and expression levels of various proteins were determined by Western blot analysis of cytosolic extracts on probing with specific antibodies. IMMU-114–mediated apoptosis is caspase-independent, as observed by unchanged levels of caspase-3 and -9.