IFN treatment enhances cleavage of caspase-8 induced by the agonistic anti-Fas antibody, but not by TRAIL. (A) Western blot analysis of caspase-8 activity. U-266-1970 cells were treated with IL-6 (20 U/mL) alone or in combination with 1000 U/mL IFN-α or 1000 U/mL IFN-γ for 96 hours, followed by a 6-hour incubation with agonistic anti-Fas antibody CH-11, recombinant soluble Apo2L/TRAIL (TRAIL), or isotype-specific control IgM. Whole cell extracts were prepared and Western blot analysis was performed as described in “Patients, materials, and methods” using specific antibodies against caspase-8 and β-actin. The uncleaved, full-length caspase-8 (p57) and the cleaved and activated forms (p41/p43 and p18) of caspase-8 are indicated. One representative experiment of 3 performed is shown. (B) Colorimetric assay of caspase-8 activity. U-266-1970 cells were treated with IL-6 (20 U/mL) alone or in combination with 1000 U/mL IFN-α or 1000 U/mL IFN-γ for 96 hours, followed by a 6-hour incubation with agonistic anti-Fas antibody CH-11, recombinant soluble Apo2L/TRAIL (TRAIL), or isotype-specific control IgM. The cells were then analyzed using the BD ApoAlert Caspase Colorimetric Assay kit. The bars represent the measured absorbance after 2 hours of incubation with the chromophore-coupled caspase-8 substrate. The graph shows one representative experiment performed in duplicate. Mean ± SD (n = 2). The asterisks indicate significant differences to the corresponding control values (P < .05).