Plinabulin-induced apoptosis in MM cells is associated with activation of caspases and JNK. (A) MM.1S, MM.1R, and RPMI-8226 MM cells were treated with plinabulin (8nM) for 48 hours and harvested, and total protein lysates were subjected to Western blot analysis with the use of antibodies against PARP, caspase-3, caspase-8, caspase-9, or GAPDH. TL indicates total length; CF, cleaved fragment. Blots shown are representative of 2 independent experiments. (B) MM.1S and MM.1R MM cells were treated with plinabulin (8nM) for 48 hours and harvested, and total protein lysates were subjected to Western blot analysis with antibodies against pJNK or GAPDH. Blots shown are representative of 2 independent experiments. (C) MM.1S, MM.1R, and RPMI-8226 MM cells were pretreated with the biochemical inhibitor of JNK (SP600125; 20μM for 30 minutes), followed by plinabulin treatment (8nM, 48 hours). After incubation, cell death was measured with the Trypan Blue assay (n = 3; P < .05). (D) MM.1S cells were transfected with 100nM siRNA JNK I or JNK II or scrambled siRNA with the use of the cell line Nucleofactor Kit V solution (Amaxa Biosystems/Lonza) for 72 hours, and protein expression of JNK-I or JNK-II was examined by immunoblotting with antibodies specific for JNK I and II. (E) Transfected MM.1S cells were treated with plinabulin (8nM, 48 hours), and cell viability was measured with the MTT assay (n = 3; P > .05).