Figure 4

Effects of DR5/Fc and DR5 siRNA on AFMC-induced DR5 expression and AFMC/TRAIL-induced apoptosis. Figure 4A: A549 cells were transfected with LacZ siRNA or siRNA against DR5 followed by treatment with 1.0 μmol/L AFMC for 24 h. DR5 levels were measured by Western blotting. β-actin was used as a loading control. Figure 4B: A549 cells were preincubated with 1.0 μg/mL DR5-specific blocking chimera antibody or transfected with siRNAs, incubated for 24 h, and then treated with or without 1.0 μmol/L AFMC for 30 min followed by treatment with 30 ng/mL TRAIL for 24 h. The frequency of cells in the sub-G1 phase was measured using flow cytometry. Data are shown as mean ± S.D. (n = 3). aP < 0.05 vs. 0.2% DMSO; bP < 0.05 vs. combined treatment. Figure 4C: A549 cells were treated as described above. Enzymatic activity of caspase-3 was determined by incubation of 20 μg of total protein with 200 μM of Ac-DEVD-pNA in 100 μL of assay buffer for 2 h at 37°C. pNA release was monitored spectrophotometrically at 405 nm. Data are shown as mean ± S.D. (n = 3). aP < 0.05 vs. 0.2% DMSO; bP < 0.05 vs. combined treatment. Figure 4D: A549 cells were treated as described above. Enzymatic activity of caspase-8 was determined by incubation of 20 μg of total protein with 200 μM of Ac-IETD-pNA in 100 μL of assay buffer for 2 h at 37°C. pNA release was monitored spectrophotometrically at 405 nm. Data are shown as mean ± S.D. (n = 3). aP < 0.05 vs. 0.2% DMSO; bP < 0.05 vs. combined treatment.