Figure 4.

Treatment with em08red triggered the intrinsic apoptotic pathway and suppressed the antiapoptosis molecules.

Notes: (A) Cells were treated for 24, 48, and 72 hours and then harvested for detection of the annexin V-positive population by flow cytometry. (B) FaDu cells were treated with em08red (10 μM) for 24 hours, and intracellular caspase-3, caspase-8, and caspase-9 activity was detected using a CaspGLOW™ fluorescein active caspase staining kit. Control cells were incubated with vehicle only (0.1% dimethyl sulfoxide). The data are presented as the fold increase in treated samples and the asterisk indicates a significant difference between the treated group and the control group (P<0.05). (C) Treated FaDu cells were harvested and lyzed at the indicated times. Equal amounts of total cell lysates were subjected to Western blot analysis. Antibodies specific for cleaved caspase-3, caspase-8, or caspase-9 were used in the experiment. Actin expression served as the loading control. (D) Total lysates extracted from treated FaDu cells were resolved by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. After transferring to the membranes, antibodies specific for Bcl-2, Bcl-x, Bak, and actin were reacted with the indicated membranes. Actin expression served as the loading control. All Western blotting images were acquired on an LAS-4000 biomolecular imager.