MLN4924 induces cytotoxicity in human MM cell lines. (A) HMCLs were treated with increasing concentrations of MLN4924 for 48 or 72 hours, respectively. MTT assays were performed to assess growth inhibition of MLN4924. Data represent mean ± SD derived from 3 independent experiments. (B) HMCLs were cultured with increasing doses of MLN4924 (0.625-5 µM) for 24 hours and assessed for apoptosis using flow cytometry for annexin V/PI. (C) HMCLs were treated with MLN4924 for 24 hours and cell lysates were assessed by western blotting for PARP, cleaved caspase 3, cleaved caspase 9, and cleaved caspase 8. (D) HMCLs were exposed to MLN4924 for 24 hours, before cell collection, cells were incubated with or without 100 nM bafilomycin A1 for 2 hours. HMCL, human myeloma cell line.