Figure 8: Neddylation-mediated Smurf1 activation promotes the progression of colorectal cancer.
(a) Smurf1 WT or C426A, C530A, C699A mutant vectors were each stably transfected into HCT116 cells that were depleted of Smurf1 using a Lentivirus-coupled shRNA against Smurf1. The expression of Smurf1 was detected by western blot to determine the efficiency of depletion (lane 2) and to compare expression levels among the overexpression groups (lanes 3–6). (b) Volume of xenograft tumours derived from HCT116 cells (5 × 106 per mouse) subcutaneously inoculated into the right flank of the mice (n=6 in each group). The transplanted tumours were removed and photographed. (c) Six nude mice were injected subcutaneously for each of the indicated stable cell lines. Tumours were isolated, and their weights were measured. (d) TV was measured using a caliper at the indicated time points. Data are shown as the mean±s.d. (e) The in vitro cell migration assay was performed in transwell plates. Representative results from three independent experiments are shown. (f) An MTS assay was performed. Cell proliferation was assessed every 24 h. Data are presented as the mean±s.d. Results are from a representative experiment performed in triplicate. (g) Working model of Nedd8-mediated conjugation on the activation of Smurf1 ubiquitin ligase.
