Molecular events associated with exposure to carfilzomib. (A) ANBL-6 cells were exposed to a pulse of 100 nM carfilzomib, bortezomib, or vehicle (Veh) control and allowed to recover for 8 or 24 hours. Cellular lysates (30 μg per reaction) were incubated with 40-μM fluorogenic substrates specific for caspase-3, caspase-8, and caspase-9. Results are expressed as fold relative fluorescence units over DMSO control. (B) ANBL-6 (2 × 10⁴ cells per reaction) were pretreated for 20 hours with caspase-3 (C-3)-, caspase-8 (C-8)-, and caspase-9 (C-9)-specific inhibitors, a negative control (Neg), a pan-caspase inhibitor (Pan), or no inhibitor (−), followed by a 1-hour pulse with 100 nM carfilzomib. Fresh media containing caspase inhibitors were then added, and cellular proliferation was determined after a 24-hour recovery period. Data were expressed as percent inhibition compared with vehicle (DMSO) controls. (C) Carfilzomib induces depolarization of mitochondria. The fluorescent shift of the JC-1 cationic dye from cytosol (green fluorescence) to mitochondria (red fluorescence) in live cells was analyzed by flow cytometry in ANBL-6 cells pulsed with 100 nM carfilzomib (Q1: red fluorescence; Q2: red and green fluorescence; Q3: green fluorescence). (D) ANBL-6 cells treated for 1 hour with 100 nM carfilzomib were subjected to centrifugal cellular fractionation into the cytosolic fraction and HMF that included mitochondria. Release of cytochrome c and Smac from the mitochondria was assessed by Western blot. Cox II, an intramitochondrial protein, was used as a control. WCEs were monitored as a control for protein expression. (E) Western blot analysis for the phosphorylation of JNK (ie, activated JNK) and cleavage of PARP in RPMI 8226 cells, which were pulsed with 100 nM carfilzomib and allowed to recover for the indicated time periods. HSC-70 was used as a loading control. To determine whether abrogation of JNK signaling through c-Jun affects carfilzomib's antiproliferative and proapoptotic action, ANBL-6 cells (2 × 10⁴ per well) were infected with DN-c-Jun adenovirus for 24 hours, followed by addition of 100 nM pulse with carfilzomib. (F) Cellular growth was assessed using the WST-1 reagent. (G) Apoptosis was measured by DNA fragment production. Error bars for panels A, B, F, and G are SD.