Fig. 4. Depletion of OTUD6A leads to destabilization of c-Myc and inhibits prostatic tumorigenesis in vitro and in PrCa xenograft mice.

A Depletion of OTUD6A led to reduced protein levels of c-Myc in 22Rv1 and C4-2 PrCa cells. 22Rv1 and C4-2 cells were infected with shControl or shOTUD6A virus, and selected with puromycin for 72 h, followed by IB assay for indicated proteins. B, C Depletion of OTUD6A reduced the half-life of c-Myc protein in 22Rv1 cells. Cells were infected with shControl or shOTUD6A virus and selected with puromycin for 3 days, followed by transient transfection with EV or shRNA-resistant Flag-OTUD6A constructs (Flag-OTUD6A-R). These cells were then treated with 200 µg/ml of CHX for indicated time before harvest, followed by IB assay for indicated proteins (B) and quantification (C). D Depletion of OTUD6A suppressed cell proliferation of 22Rv1 and C4-2 PrCa cells. 22Rv1 and C4-2 cells as in Fig. 4A were subjected to growth curve analysis. E Representative images showed the colony growth of human PrCa cells after depletion of endogenous OTUD6A. F, G Depletion of OTUD6A suppressed the colony growth of 22Rv1 and C4-2 cells. The cells as in Fig. 4A were subjected to colony formation assay. ***p < 0.001. H Depletion of OTUD6A using CRISPR/Cas9 led to reduced protein levels of c-Myc in 22Rv1 and C4-2 cells. Immunoblot of WCL derived from 22Rv1 cells with OTUD6A knockout by CRISPR/Cas9. I–K Depletion of OTUD6A by CRIPSR/Cas9 suppressed the colony growth of 22Rv1 and C4-2 cells. OTUD6A knockout cells as in Fig. 4H were subjected to colony formation assay (I) and quantification (J, K). **p < 0.01, ***p < 0.001. L, M Depletion of OTUD6A suppressed the xenograft tumor growth of 22Rv1 cells. OTUD6A-WT and OTUD6A-KO cells (as in Fig. 4H) were subcutaneously injected into 6-week-old male null mice (N = 6). The tumor mass was imaged (L) and weighed (M) at end of the experiment. ***p < 0.001. The relevant raw data are provided in Supplementary Materials.