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. 2017 Feb 1;8(16):26718–26731. doi: 10.18632/oncotarget.14957

Figure 6. Overexpression of TRIP13 induces proteasome-mediated MAD2 degradation through Akt pathway.

Figure 6

A. Western blot of MAD2 in human TRIP13 overexpressing myeloma ARP1, OCI-My5, H929 cells and HEK293T cells (OE) as well as their controls transfected with empty vectors (EV). B. Realtime PCR analysis of MAD2 mRNA in human TRIP13 overexpressing myeloma ARP1, OCI-My5, H929 cells and HEK293T cells (TRIP13-OE) as well as their controls transfected with empty vectors. Results from 3 independent experiments are shown (OE vs. Empty Vector, p > 0.05). C. Western blot of MAD2 in wild type ARP1 (ARP1-WT) cells treated with proteasome inhibitor MG132 and Bortezomib for indicated period of time. D. Western blot of MAD2 in TRIP13 overexpressing ARP1 cells (ARP1-OE) and control cells transfected with empty vectors (ARP1-EV) treated with proteasome inhibitor MG132 for indicated period of time. Bands of MAD2 have been quantified. Results from 3 independent experiments are shown (*p < 0.05). E. Western blot of MAD2 in TRIP13 knockdown ARP1 cells (ARP1-ShRNA) and the scrambled control (ARP1-Scr) treated with proteasome inhibitor MG132 for indicated period of time. Bands of MAD2 have been quantified and data. Results from 3 independent experiments are shown (*p < 0.05). F. Co-immunoprecipitation (Co-IP) demonstrated ubiquitination of MAD2 was increased in TRIP13 overexpressing HEK293T cells (293T-OE) compared to control cells transfected with empty vectors (293T-EV). G. Western blot of signaling pathways including Akt, P38 MAPK, P44/42 MAPK in TRIP13 overexpressing ARP1 cells (OE) and control cells transfected with empty vectors (EV). H. Western blot of MAD2 in TRIP13 overexpressing ARP1 cells treated with10 μMPI3K/Akt inhibitor LY294002 for 12 h or 10 μMMG132 for 4 h independently, or with the combination of LY294002 and MG132 (LY+MG). Bands of MAD2 have been quantified. Results from 3 independent experiments are shown (*p < 0.05). I. TRIP13 overexpressing ARP1 cells (TRIP13-OE) were pretreated with or without 10 μMLY294002 (LY) for 2 hours and then cells were treated with 5 nM bortezomib (Bor) for 24 h. Cell viability assay was performed. Results from 3 independent experiments are shown (*p < 0.05).