ABC294640 enhanced c-Myc proteasome degradation. (A) ABC294640 downregulated c-Myc and pS6 protein expression. MM cells (OPM1, RPMI-8226, MM.1S, JK6L, and U266) were treated with 30 µM of ABC294640 (A) or DMSO (D) for 16 hours, and whole cell lysates were prepared and analyzed for c-Myc and pS6 expression by western blot analysis. β-actin was used as the loading control. (B) ABC294640 increased c-Myc protein degradation. OPM1 cells were treated with 30 µM of ABC294640 or DMSO for 3 hours and then cyclohexamide (100 µg/mL) was added. Cells were collected at each hour after cyclohexamide treatment, and whole cell lysate was prepared and analyzed for c-Myc expression by western blot analysis. β-actin was used as the loading control. The graph represents the quantification of western blots using ImageJ. (C) Proteasome inhibitor (MG132) prevented the degradation of c-Myc by ABC294640. OPM1 cells were treated with DMSO control buffer or MG132 (1 µM) for 1 hour, followed by treatment with DMSO or 30 µM of ABC294640 for an additional 6 hours. Whole cell lysate was prepared and analyzed for c-Myc expression by western blot analysis. Data shown in the figure were representative of at least 2 separate sets of experiments.