Fig. 2: BIX-01294 induces downregulation of XIAP expression at the post-translational level.

a Caki cells were treated with the indicated concentrations of BIX for 24 h. The mRNA levels of XIAP and actin were determined by RT-PCR (upper panel) and quantitative PCR (qPCR, lower panel). The level of actin was used as a loading control. b Caki cells were treated with or without 10 μM BIX in the presence of 20 μg/ml cyclohexamide (CHX) for the indicated time periods. The protein levels of XIAP and actin were determined by western blotting. The level of actin was used as a loading control (upper panel). The band intensity of the XIAP protein was measured using ImageJ (public domain JAVA image-processing program; http://rsb.info.nih.gov/ij, lower panel). c Caki cells were pretreated with 0.5 μM MG132 for 30 min and then treated with 10 μM BIX for 24 h. The protein levels of XIAP and actin were determined by western blotting. The level of actin was used as a loading control. d Caki cells were treated with 10 μM BIX or MG132 (as a positive control) for the indicated time periods or 6 h (MG132). The cells were lysed, and the proteasome activity was measured, as described in the Materials and methods section. e Caki cells were transiently transfected with pEBB/XIAP or vector plasmid. Twenty-four hours after transfection, cells were treated with 50 ng/ml TRAIL in the presence or absence of 10 μM BIX for 24 h. Apoptosis was analyzed as a sub-G1 population by flow cytometry. The protein levels of PARP, XIAP, and actin were determined by western blotting. The level of actin was used as a loading control (lower panel). The values in a, d, and e represent the mean ± SD from three independent samples; *p < 0.05 compared with the control