Figure 3.

Association between caspase-8 expression and conatumumab sensitivity in EWS. (A) Analysis of EWS cell lines for the expression of proteins relevant to DISC and its associated initiator caspases 8 and 10. (B) Sensitive Rh1 and KAD, as well as resistant A4573, cells were treated with conatumumab+anti-Fc for the indicated time. The cell lysates were analysed for the cleavage of caspase-8, caspase-3, caspase-10, and PARP by immunoblots. (C) Ectopic expression of caspase-8-GFP. Conatumumab-resistant A4573 cells were transiently transfected with CASP8 or caspase-defective CASP8^mt (C360S) expression plasmids for 2 days. Cell lysates were analysed for caspase-8-GFP expression with immunoblot, using the sensitive Rh1 cells as a reference for caspase-8. (D) Ectopic expression of caspase-8 sensitises A4573 cells to CONA+anti-Fc induced cell death. After transient transfection with CASP8 or caspase-defective CASP8^mt (C360S) expression plasmids for 2 days, the cells were then treated with CONA+anti-Fc for 3 days and analysed for cell viability. The sensitive Rh1 cells were used as a reference.