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. 2020 Aug 21;12(9):2363. doi: 10.3390/cancers12092363

Figure 3.

Figure 3

DR5 stabilization contributes to the sensitization effect of IITZ-01 on TRAIL-induced apoptosis in Caki-1 cells. (a,b) Caki-1 cells were incubated with 1 μM IITZ-01 for indicated time periods. The protein and mRNA levels were determined by Western blotting (a) and RT-PCR (upper panel)/qPCR (low pane), respectively (b); (c) Caki-1 cells were transiently transfected with DR5(-605) or DR5(SacI) promoter and incubated with 1 μM IITZ-01 for 18 h. The cells were lysed and the luciferase activity was measured as a described in a Material and Methods; (d) Caki-1 cells were incubated with 1 μM IITZ-01 for 18 h, washed with PBS, and then treated with 20 μg/mL cycloheximide (CHX) and/or 1 μM IITZ-01 for the indicated time periods. The band intensity of the DR5 protein was measured using ImageJ; (e) Caki-1 and ACHN cells were transiently transfected with control (Cont) siRNA or DR5 siRNA, and then incubated with 1 μM IITZ-01 and 50 ng/mL TRAIL for 18 h. The apoptotic population (sub-G1) and protein levels were analyzed by flow cytometry (e) and Western blot (d,e). The values in graph (b,c,e) represent the mean ± SD of three independent experiments. * p < 0.05 compared to the IITZ-01 plus TRAIL in control siRNA.