Cell death induced by plinabulin depends on JNK as well as caspases. (A) MM.1S, MM.1R, and primary patients cells were pretreated with PAN caspase inhibitor Z-VAD-FMK (40μM, 2 hours) followed by plinabulin treatment (8nM, 48 hours). After the desired time point cell death were measured with the Trypan Blue assay (n = 2; P > .05). (B) MM.1R cells were treated with higher doses of plinabulin (20nM) for 1, 6, 16, and 24 hours, and Western blot analyses were performed with antibodies against pJNK, caspase-3, caspase-8, caspase-9, or GAPDH. CF indicates cleaved fragment. Results were representative of 2 independent experiments with similar results. (C top) MM.1R cells were treated with PAN caspase inhibitor for 2 hours, and then plinabulin (8nM) was added for an additional 48 hours. Protein lysate was prepared; phosphorylation of JNK was checked with Western blot analysis. (C bottom) MM.1R cells were pretreated with JNK inhibitor SP600125 (20μM, 2 hours), and plinabulin (8nM) was added for another 48 hours. After 48 hours protein lysate was prepared and cleaved caspase-3 expression was checked with Western blot analysis. (D) MM cell line MM.1S was pretreated with SP600125 (20μM) for 2 hours, and plinabulin (8nM) was added for an additional 16 hours followed by analysis for histone H3 phosphorylation with the use of flow cytometry. Data are representation of 3 independent experiments.