Figure 3.

DR-mediated cell death in the presence of the IAP antagonist uses caspases and caspase-independent signaling pathways. (A) HaCaT cells were pre- or co-stimulated with 10 µM zVAD-fmk for 1 h and 100 nM of the IAP antagonist for 30 min. Subsequently, cells were stimulated with the indicated concentration of TRAIL or CD95L. The viability of cells was analyzed by crystal violet assay 18–24 h later as indicated in Materials and methods. Mean + SEM for three (TRAIL) or six (CD95L) independent experiments is shown. (B) HaCaT cells were either pretreated with 10 µM zVAD-fmk for 1 h or 100 nM of the IAP antagonist for 30 min. Cells were subsequently stimulated with 5 U/ml CD95L for 4 or 24 h. 5 µg/ml Hoechst 33342 and 5 pM SYTOX green were added for 15 min at 37°C, immediately followed by transmission (left) or fluorescence (right) microscopy. One of two independent experiments is representatively shown. (C) Stable knockdown of RIP1 in HaCaT cells was performed as indicated in Materials and methods and controlled by Western blot analysis for RIP1. Reprobing of the membrane with β-tubulin antibodies served as a control for protein loading. MM, molecular mass. (D) Transduced HaCaT cells as shown in C were prestimulated for 30 min with 100 nM of the IAP antagonist or diluent alone and subsequently stimulated with the indicated concentrations of TRAIL or CD95L for 18–24 h followed by crystal violet assay. Mean ± SEM of three (TRAIL) or four (CD95L) independent experiments is shown. (E) Transduced HaCaT cells, as described in C, were preincubated with 100 nM of the IAP antagonist (Ant) for 30 min and then stimulated with 0.5 U/ml CD95L. After 24 h, colony formation was assayed as indicated in Materials and methods. One of four representative independent experiments is shown. Bar, 10 µm.