Figure 4.

RanBP3 is a direct target of NU2058 and essential for the anticancer effect of NU2058 in CRC. A,B) Cell viability assay (A) and colony formation assay (B) were performed to determine the effect of NU2058 (0 and 10 µm) on the proliferation of RanBP3‐deficient cells. C) Co‐IP showing that the interaction between RanBP3 and β‐catenin was increased in the presence of NU2058. D) SPR assay showing the binding of NU2058 to the RanBP3 protein. E) The binding sites between NU2058 and RanBP3 were predicted by molecular docking. F) Schematic diagram of RanBP3 protein mutations. G,H) RanBP3‐WT, RanBP3‐Mut#1, RanBP3‐Mut#2, or RanBP3‐Mut#3 were expressed in RanBP3‐deficient cells, and the cells were then subjected to WST‐1 and colony formation assays. I) Tumor volumes and images showing that the anticancer bioactivity of NU2058 was abolished in tumor xenografts established from RanBP3‐deficient cells compared with those established from control cells (n = 6). J) Western blotting analysis of cyclin D1, CDK4, c‐Myc, and actin expression in the tumor xenografts. K) The Ki‐67 proliferation index was detected in NU2058‐treated tumor xenografts by immunohistochemistry (n = 3). Bars, SD; *p < 0.05; **p < 0.01; ***p < 0.001.