Figure 5.

DR5 contributes to bortezomib-induced TRAIL sensitization. (A) Effects of bortezomib on the expression of DR4 and DR5 in SNU-216. Cells were treated with TRAIL and/or bortezomib for 24 h and 48 h, and expression of DR4 and DR5 was examined by western blot analysis. (B) Surface expression of DR4 and DR5 in SNU-216 upon 24 h expose to TRAIL in the presence or absence of bortezomib, was analyzed by flow cytometry. (C) Lentivirus of scramble control or DR5 shRNA was transduced into SNU-216, and then selected with puromycin (2.5 µg/ml) for a week. Cell viability of scramble and DR5-silenced cells upon TRAIL and/or bortezomib treatment for 24 h and 48 h was measured by MTT assay and relative cell viability was calculated against untreated control. Results shown are means ± SE of three independent experiments. (D) Reduced Annexin V binding in the DR5-silenced cells treated with TRAIL and/or bortezomib was analyzed by flow cytometry. Lower panel shows fractions of Annexin V positive and negative cells, respectively. (E) Expression of DR5 and activation of caspases in the DR5-silenced cells treated with TRAIL and/or bortezomib were analyzed by western blot. (F) Expression of DR5 in SNU-216 was knocked down by transfection of DR5 siRNA. DR5-silenced cells by siRNA transfection were treated with TRAIL and/or bortezomib for 24 h and 48 h and cell viability was measured by MTT assay. Relative cell viability was calculated against untreated control and shown are the means ± SE of three independent experiments. (G) Expression of DR5 and activation of caspases in the DR5-silenced cells were examined by western blot analysis. The siRNA transfected cells were treated with TRAIL and/or bortezomib for 24 h. All cells (A~G) were treated with TRAIL at 12.5 ng/ml and/or bortezomib (22.75 nM) for indicated time period. * for P< 0.05 and 'ns' for P>0.05 calculated from Student's t-test (C, D and F). Results shown are representatives of triple experiments (A, B D, E and G).