Figure 3.

The downregulation of c-FLIP(L) and Mcl-1 by thioridazine is associated with the induction of TRAIL-mediated apoptosis. (a) Caki cells were treated with 10 μM thioridazine for the indicated time periods. (b) Caki cells were treated with or without 10 μM thioridazine (Thio) in the presence of CHX (20 μg/ml) for the indicated time periods. (c and d) Vector cells (Caki/vector), c-FLIP(L)-overexpressed cells (Caki/cFLIP (L)), and Mcl-1-overexpressed cells (Caki/Mcl-1) were treated with 50 ng/ml TRAIL in the presence or absence of 10 μM thioridazine for 24 h. The level of apoptosis was analyzed by the sub-G1 fraction using flow cytometry. The protein and mRNA expression levels of c-FLIP(L) and/or Mcl-1 were determined by western blotting (a, b, c, and d) and RT-PCR (a), respectively. The level of actin was used as a loading control. The band intensities of c-FLIP(L) and Mcl-1 protein were measured using the public domain JAVA image-processing program ImageJ (b, lower panel). *P<0.001 compared with the thioridazine plus TRAIL-treated Caki/vector cells. The data represent three independent experiments