Figure 4.

Thioridazine increases the proteasome activity. (a) Caki cells were pretreated with 1 μM MG132 or 5 μM lactacystin for 30 min and then treated with 10 μM thioridazine for 24 h. (b) Caki cells were treated with 10 μM thioridazine or MG132 (as a positive control) for the indicated time periods. The cells were lysed, and the proteasome activity was measured as described in the Materials and Methods section. (c) Caki cells were transfected with proteasome sensor vector (ZsProSensor-1) and treated with 10 μM thioridazine or MG132 (as a positive control) for the indicated time periods. The proteasome activity was analyzed using FACS. (d) Caki cells were treated with 10 μM thioridazine for the indicated time periods. The protein expression levels of PSMA5, PSMD4/S5a, and actin were determined by western blotting. The level of actin was used as a loading control. (e) Caki cells were pretreated with 1 μM MG132 or 5 μM lactacystin for 30 min and then treated with 10 μM thioridazine and 50 ng/ml TRAIL for 24 h. The level of apoptosis was analyzed by measuring the sub-G1 fraction by flow cytometry (left panel). The protein expression levels of PARP, c-FLIP(L), Mcl-1, and actin were determined by western blotting. The level of actin was used as a loading control. The values in (b and e) represent the mean±S.D. from three independent samples. *P<0.001 compared with control. ^#P<0.001 compared with thioridazine plus TRAIL. The data represent three independent experiments