Fig. 5. OTUD6A promotes prostatic tumorigenesis by deubiquiting and stabilizing c-Myc oncoprotein.

A Alignment the conserved TXXXS consensus of c-Myc among species. B The non-degradable version of the c-Myc-T58AS62A (2A) mutant did not interact with OTUD6A. Immunoblot of Flag-immunoprecipitants, HA immunoprecipitants and WCL derived from HEK293T transfected with Flag-OTUD6A and HA-tagged constructs. C OTUD6A removed the poly-ubiquitin chain from c-Myc protein depending on the phosphorylation events. Immunoblot of Ni-NTA pull-downs and WCL derived from HEK293T cells transfected with His-Ub, Flag-OTUD6A and HA-tagged constructs. Cells were pretreated with 30 μM of MG132 for 6 h before harvest. D, E Ectopic expression of OTUD6A extended the half-life of WT c-Myc protein, but not of the 2A mutant. Immunoblot of WCL derived from HEK293T cells transfected with Flag-OTUD6A and HA-tagged constructs. The cells were treated with CHX (200 µg/ml) for indicated time before harvest. F Depletion of endogenous OTUD6A reduced the protein level of WT c-Myc, but not of the c-Myc 2A mutant. Immunoblot of WCL derived from 22Rv1 cells stably expressed with either HA-c-Myc-WT or HA-c-Myc-2A, with or without depletion of OTUD6A by shRNA. G Depletion of OTUD6A reduced the cell growth of 22Rv1 that stably expressed WT c-Myc, but not of the cell expressing 2A mutant. Cells as in Fig. 5F were subjected to growth curve assay. ***p < 0.001. H, I Depletion of OTUD6A reduced the tumorigenesis ability of 22Rv1 in a xenograft assay. Cells as in Fig. 5F were subcutaneously injected into 6-week-old male null mice (N = 6). The tumor mass was imaged (H) and weighed (I) at end of the experiment. ***p < 0.001. The relevant raw data are provided in Supplementary Materials.