Figure 6

Caspase8-depleted ovarian cancer cells show evidence of cell death by necroptosis. (a) Western analysis was performed on cell lysates obtained from Ovcar3 and Caov3 cells expressing control or Caspase8 shRNA #2, after treatment with IKKβ inhibitor (2.5 μM) and/or TNFα (10 ng/ml) for 18 h. Protein levels of Caspase8, RIPK1 (uncleaved, 78 kDa), MLKL and cleaved PARP are shown. GAPDH was used as loading control (b) Western analysis was performed on cell lysates obtained from Ovcar3 cells expressing control or Caspase8 shRNA #2, after treatment with IKKβ inhibitor (2.5 μM), SMAC-mimetic (birinapant 200 nM) and/or TNFα (10 ng/ml) for 18 h. Protein levels of Caspase8, RIPK1 (uncleaved, 78 kDa), MLKL, cIAP1 and cleaved PARP are shown (upper). β-Tubulin was used as loading control. (c) Ovcar3 cells expressing either control or Caspase8 shRNA #2 were transiently transduced with control or RIPK1 siRNA. (d) Ovcar3 cells described in c were exposed to IKKβ inhibitor with or without TNFα stimulation, in the presence of apoptosis inhibitor (ZIETD) or necroptosis inhibitor (NEC1). Cells were treated for 18 h with TNFα (10 ng/ml), IKKβ inhibitor (2.5 μM), SMAC-mimetic (birinapant 200 nM) and/or ZIETD (25 μM) or NEC1 (25 μM) as indicated. Viability was assessed by XTT assay. Results are expressed as average±S.E.M., n=8, *P<0.01 based on t-test, and shown as percent change from no treatment control.