Figure 3

DR5 had an important role in the synergistic effect of CK and TRAIL. HCT116 cells were treated with CK at the indicated doses for 24 h. The anti-apoptotic (a), pro-apoptotic proteins (b) and DR4/ DR5 proteins (c) were analyzed by western blotting. Actin was used as a protein loading control. The DR4 and DR5 mRNA levels were analyzed by RT-PCR after HCT116 cells were treated with CK at the indicated doses for 24 h. GAPDH was used as an internal control to show equal RNA loading (d). The cell surface DR4 and DR5 expression was tested by flow cytometry followed by CK (50 μM; 24 h) treatment (e). HCT116 cells were transfected with control siRNA or DR5 siRNAs. After treatment with CK for 24 h, whole-cell extracts were prepared and analyzed by western blotting (f). The resultant cells were exposed to 50 μM CK for 24 h and then co-treated with or without 25 ng/ml TRAIL for 24 h. Cells were stained with PI and cell death (as indicated by sub-G1 DNA content to the left of the G1 peak) was measured by FACS (g). Error bars in (g) represent the S.D. (N=3 independent experiments). *P<0.05, compared with DMSO group (Student’s t-test, two tailed)