Figure 4.

Knockdown of endogenous c-FLIP_S in several cell lines enhances apoptosis induction by TNF-α. (A) To examine the role of c-FLIP_S in TNFR1-mediated apoptosis, PC3 cells were transfected with three different siRNA oligonucleotides designed to knock down c-FLIP_S. The level of c-FLIP_S protein was inhibited completely by C2 and C3. In further studies, we used C3 oligonucleotide as an siRNA. (B) The indicated siRNA-transfected cells were treated with TNF-α (5 ng/ml) for 24 h and extracted for immunoblotting with phospho-specific antibodies to JNK and actin. (C) The indicated siRNA-transfected cell lines were treated with TNF-α (5 ng/ml) for 24 h before harvesting for flow cytometric analysis. This experiment was performed independently three times. (D) The indicated siRNA-transfected cell lines were treated with TNF-α (5 ng/ml) for 24 h and extracted for immunoblotting with anti-FLIP_S antibody. β-Actin was used as a loading control.