Caspase 3 activation was caspase 8 dependent in normal (JY), FA-C (HSC536N), and FANCC-corrected (HSC536N FANCC/neo) cells.

(A) Immunoblot for caspase 8 on lysates from HSC536N and HSC536N FANCC/neo cells treated with 1.0 ng/mL IFN-γ and 100 ng/mL anti-fas antibody for 120 minutes shows the 12-kd cleavage product, demonstrating caspase 8 activation by this treatment. (B) Fluorogenic assays for caspase 3 on lysates from IFN-γ and anti-fas antibody-treated JY and HSC536N cells revealed high levels of induced caspase 3 activation. The caspase 8 inhibitor, ac-IETD-FMK (50 μmol/L), abrogated activation of caspase 3. The graph shown is representative of 3 experiments. No effect of ac-IETD-FMK was detected on caspase 3 activation in the absence of anti-fasantibody and IFN-γ. (C) Immunoblot of lysates from JY and HSC536N cells pretreated for 30 minutes with Z-IEDT-FMK (50 μmol/L) (indicated by I). Because DMSO was required to dissolve ac-IETD-FMK, control samples were pretreated with DMSO alone (indicated by C). Cells were then treated with 100 ng/mL anti-fas antibody and 1.0 ng/mL IFN-γ for 60 minutes.