Cell death and molecular changes associated with IPSI-001. (A) WST-1 cytotoxicity assay in ANBL-6 cells treated for 18 hours with bortezomib or IPSI-001. The data shown are the mean plus or minus SD from 3 independent experiments. (B) Flow cytometric analysis was performed after annexin V staining in RPMI 8226 cells treated for 24 hours with the indicated concentrations of IPSI-001, with each experiment performed in duplicate. Error bars indicate SD. *P < .05 comparing treated cells over vehicle control. (C) Representative results from 2 independent experiments of a colorimetric activity assay for caspases in RPMI 8226 myeloma cell lysate (100 μg) treated for up to 24 hours with 50 μM IPSI-001. The data shown are the mean plus or minus SD from duplicate experiments. *P < .05 for fold caspase activity over baseline. (D) Western blot analysis for the late-stage apoptotic marker PARP, where the cleaved fragment (CF) represents apoptotic cells compared with the full-length (FL) PARP, which is present in healthy, nonapoptotic ANBL-6 cells. HSC-70 levels were used as a loading control. (E) Protein expression of Bax and IκBα in RPMI 8226 cells after incubation with 50 μM IPSI-001 for 24 hours. Actin levels were used as a loading control. (F) Protein expression of phosphorylated JNK (phospho-JNK) and total JNK in RPMI 8226 cells treated with 25 or 50 μM IPSI-001 for 24 hours.