The effect of up-regulation of DR5 by garcinol on TRAIL sensitization. (A) Caki cells were treated with 2 μM of garcinol for 3–24 h. Western blot (upper) and RT-PCR (lower) showing the DR5 and actin; (B) After garcinol treatment for 12 h, Caki cells were treated with 20 μg/mL cycloheximide (CHX) in the presence or absence of 2 μM garcinol for 1–12 h. Western blot showing the DR5 and actin. The band intensity was calculated using Image J; (C) Caki cells were treated with 2 μM garcinol for 3–24 h. Western blot showing the PSMA5, PSMD4/S5a, Itch, Cbl and actin; (D) For DR5 surface staining, cells were treated with 2 μM garcinol for 24 h and the cell surface expression level of DR5 was determined by flow cytometry as mentioned in a material and methods; (E) Caki cells were transfected with control or DR5 siRNA, and then cells were treated with 2 μM garcinol plus 50 ng/mL TRAIL for 24 h. Levels of apoptosis were assessed by flow cytometry, and western blot showing the PARP, DR5 and actin. * p < 0.01 compared to the control. ** p < 0.01 compared to the garcinol plus TRAIL-treated control siRNA.