Figure 6

AFMC neither induce expression of DR5 nor enhance TRAIL-induced apoptosis in WI-38 cells. Figure 6A: WI-38 cells were treated with AFMC for 30 min and then treated with TRAIL for 24 h at the indicated concentrations. Cytotoxicity was assessed by ELISA. Columns, average of three individual experiments; bars, SD. a P < 0.05 vs. 0.2% DMSO; c P < 0.05 vs. 1.0 μmol/L AFMC. Figure 6B: WI-38 cells were treated with 1.0 μmol/L AFMC, 30 ng/mL TRAIL or the pretreated with 1.0 μmol/L AFMC for 30 min followed by 30 ng/mL TRAIL for 24 h. DNA content of the cells was analyzed by flow cytometry. Figure 6C: WI-38 cells were treated as described above. The enzymatic activity of caspase-3 was determined by incubation of 20 μg 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. Figure 6D: WI-38 cells were treated as described above. The 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). Figure 6E: WI-38 cells were treated as described above. Fragmented DNA was extracted from the treated cells and analyzed on a 2.0% agarose gel. Figure 6F: WI-38 cells were treated with the indicated concentrations of AFMC for 24 h, and DR5 levels were assessed by Western blotting. β-actin was used as a loading control.