To determine the functional significance of SUB1 expression in prostate cancer, we perturbed SUB1 levels in prostate cells and investigated the effect of this modulation on cell proliferation, migration and invasion. We used both transient RNA interference and stable knockdown strategies targeting SUB1 in aggressive prostate cancer cell lines DU145 and PC3 and hormone-responsive LnCaP and VCaP cells. The efficiency of SUB1 knockdowns were confirmed by immunoblot (Figure 3a) and qPCR (Supplementary Figures S3a–g) analyses. We observed significant decrease in cell proliferation upon transient knockdown of SUB1 compared with control cells transfected with non-T small interfering RNAs (siRNAs) (Figure 3a). Next, we tested cell motility after stable SUB1 knockdown in prostate cancer cells using wound healing assay. The efficiency of SUB1 stable knockdowns were confirmed by immunoblot (Supplementary Figure S4a). SUB1 knockdown showed a wider wound area 24 h post wound generation relative to control cells, the delayed time to heal indicating an inability of SUB1 knockdown cells to migrate (Supplementary Figures S4b and c). Additionally, SUB1 knockdown in DU145 and PC3 reduced the invasive potential of these cells as assessed by Boyden chamber Matrigel invasion assay (Figure 3b). Further we tested for colony formation after transient knockdown of SUB1 and colonies were quantified (Figures 3c and d). Taken together, these observations demonstrate the involvement of SUB1 in the proliferation, migration, invasion and colony formation of prostate cancer cells in vitro.