FIG. 4.

Effect of the nonspecific caspase inhibitor Boc-D-Fmk on apoptosis and IL-8 induction upon DR5 ligation. (A) CRT-MG cells were incubated in the absence or presence of the cell-permeable nonspecific caspase inhibitor Boc-D-Fmk (0 to 50 μM) for 3 h prior to a 24-h treatment with hrTRAIL (50 ng/ml), and cell death was evaluated by staining with Annexin-V and PI as indicated in Materials and Methods. Data are the mean and SD for duplicate samples from one experiment and are representative of three independent experiments. (B) CRT-MG cells were pretreated in the absence (second lane) or presence (third lane) of Boc-D-Fmk (50 μM) for 3 h, washed twice, and then treated with hrTRAIL (50 ng/ml) for an additional 24 h. IL-8 mRNA expression was measured by an RPA; fold induction of IL-8 is indicated. Data are representative of three experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) CRT-MG cells were stably transfected with construct IL-8p-d2EGFP as indicated in Materials and Methods. The stable reporter cells were incubated in the absence or presence of Boc-D-Fmk (0 to 50 μM) for 3 h and treated with hrTRAIL (100 ng/ml) for an additional 24 h, and then EGFP expression was examined by FACS analysis. Data are the mean and SD for duplicate samples from three independent experiments. The asterisk indicates a P value of ≤0.05. (D) Cells were incubated in the absence or presence of Boc-D-Fmk (50 μM) for 3 h and washed twice prior to a 36-h treatment with hrTRAIL (50 ng/ml), and IL-8 protein expression was measured by an ELISA. Data are the mean and SD for quadruplicate samples from one experiment. Values significantly different from the values for the hrTRAIL-treated sample without Boc-D-Fmk pretreatment are indicated by an asterisk (P ≤ 0.05). The data are representative of two independent experiments.