Figure 4
Figure 4. ABC294640 enhanced Mcl-1 proteasome degradation and increased Noxa expression. (A) ABC294640 downregulated Mcl-1 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 Mcl-1 expression by western blot analysis. β-actin was used as the loading control. Data were representative of 3 separate sets of experiments. (B) A294640 increased Mcl-1 protein degradation. OPM1 cells were treated with 30 µM of ABC294640 or DMSO for 3 hours and then cycloheximide (100 µg/mL) was added. Cells were collected at each hour after cycloheximide treatment and whole cell lysate was prepared and analyzed for Mcl-1 expression by western blot analysis. β-actin was used as the loading control. The graph represents the quantification of western blots. The western blots were quantified using ImageJ. Data were representative of 2 separate sets of experiments. (C) Proteasome inhibitor (MG132 and bortezomib) prevented the degradation of Mcl-1 by ABC294640. OPM1 cells were treated with DMSO control buffer, proteasome inhibitor MG132 (1 µM), or bortezomib (50 nM) 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 Mcl-1 expression by western blot analysis. Data were representative of 2 separate sets of experiments. (D) ABC294640 increased Noxa protein expression. OPM1, RPMI8226, MM.1S, and JK6L were treated with 30 µM of ABC294640 (A) or DMSO (D) for 16 hours, and whole cell lysates were prepared and analyzed for Noxa expression by western blot analysis. (E) ABC294640 induced Noxa gene expression. Eight MM cell lines were treated with 30 µM of ABC294640 (A) or DMSO (D) for 16 hours and RNA was isolated and analyzed for Noxa gene expression by RT-PCR. Gene expression was normalized against β-actin internal control. Graphs represented the fold change of Noxa mRNA in ABC294640-treated MM cells lines compared with DMSO-treated cells. Data shown in the figure were representative of at least 2 separate sets of experiments.

ABC294640 enhanced Mcl-1 proteasome degradation and increased Noxa expression. (A) ABC294640 downregulated Mcl-1 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 Mcl-1 expression by western blot analysis. β-actin was used as the loading control. Data were representative of 3 separate sets of experiments. (B) A294640 increased Mcl-1 protein degradation. OPM1 cells were treated with 30 µM of ABC294640 or DMSO for 3 hours and then cycloheximide (100 µg/mL) was added. Cells were collected at each hour after cycloheximide treatment and whole cell lysate was prepared and analyzed for Mcl-1 expression by western blot analysis. β-actin was used as the loading control. The graph represents the quantification of western blots. The western blots were quantified using ImageJ. Data were representative of 2 separate sets of experiments. (C) Proteasome inhibitor (MG132 and bortezomib) prevented the degradation of Mcl-1 by ABC294640. OPM1 cells were treated with DMSO control buffer, proteasome inhibitor MG132 (1 µM), or bortezomib (50 nM) 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 Mcl-1 expression by western blot analysis. Data were representative of 2 separate sets of experiments. (D) ABC294640 increased Noxa protein expression. OPM1, RPMI8226, MM.1S, and JK6L were treated with 30 µM of ABC294640 (A) or DMSO (D) for 16 hours, and whole cell lysates were prepared and analyzed for Noxa expression by western blot analysis. (E) ABC294640 induced Noxa gene expression. Eight MM cell lines were treated with 30 µM of ABC294640 (A) or DMSO (D) for 16 hours and RNA was isolated and analyzed for Noxa gene expression by RT-PCR. Gene expression was normalized against β-actin internal control. Graphs represented the fold change of Noxa mRNA in ABC294640-treated MM cells lines compared with DMSO-treated cells. Data shown in the figure were representative of at least 2 separate sets of experiments.

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