FIGURE 4.

A truncated, signaling-incapable TRAILR4 variant confers protection from TRAILR1-induced apoptosis. A, flow cytometric analysis of TRAIL receptor cell surface expression in HeLa R4ΔC cells. B, HeLaR4ΔC (diamonds) in comparison to HeLa cells (squares) were treated with serial dilutions of antibody-cross-linked sTRAIL (TRAIL) in combination with 0.5 μg/ml cycloheximide. After 24 h cell viability was determined by crystal violet staining. Shown are the mean values ± S.D. calculated from nine independent experiments each performed in triplicate. Significance was tested using two-way analysis of variance in combination with the Bonferroni post-test. ***, p < 0.001 was considered significant. Open diamonds show viability of HeLa R4ΔC cells preincubated with the pan-caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone (20 μm) before TRAIL treatment (n = 3). C, caspase cleavage in HeLa wild type cells and cells overexpressing TRAILR4. Cells were treated with 300 ng/ml antibody-cross-linked sTRAIL for the indicated times or left untreated. Total cell extracts were subjected to SDS-PAGE and Western blot analysis using antibodies directed against cleaved caspase-8 or caspase-3. Tubulin-α was used as loading control. Blots shown are representative of three independent experiments.