Activation of caspases by NF-κB inhibition.

(A) Immunoblotting analysis for caspase-9, caspase-3, and caspase-8, as well as PARP, was performed in lysates of MM.1S cells treated with SN50 (20 μM) for 0, 4, 8, or 16 hours. Treatment with SN50 induced cleavage of caspase-9, caspase-3, and PARP. In contrast, no cleavage of caspase-8 was detected upon SN50 treatment. TRAIL/Apo2L (300 ng/mL for 5 hours) induced caspase-8 cleavage, as in our prior studies,40 and served as a positive control. (B) Annexin V–PI staining was performed to quantify phosphatidylserine externalization in MM.1S cells treated with or without SN50 (20 μM) for 4 hours, in the presence or absence of specific inhibitors for caspase-3, caspase-8, and caspase-9. The caspase-9 inhibitor LEHD-FMK and the caspase-3 inhibitor DEVD-FMK partially blocked SN50-induced cell death, whereas the caspase-8 inhibitor IETD-FMK had no effect.