Fig 2: WISP1 neutralized RNF180-mediated effects on cell proliferation and apoptosis. (A) mRNA expression and protein levels of WISP1 in CRC cell lines and fetal colon cell line FHC were determined by quantitative RT-PCR and Western blot, respectively. (B) WISP1 was overexpressed in both LOVO and RKO cells, and WISP1 overexpression was confirmed. (C) WISP1 was silenced using shRNA hairpins in SW620 cells, and knockdown efficiency was validated. CRC cells were transduced with the indicated plasmids, and cell proliferation assay (D–F), apoptosis assay (G,H), and immunoblotting (I) were carried out. *P < 0.05, ***P < 0.001 compared with shNC or vector. ###P < 0.001 compared with RNF180.

Fig 3: Relationship between clinical factors and RNF180 expression in CRC. (A) The mRNA expression of RNF180 was evaluated in patient samples from 24 TCGA projects. (B) RNF180 expression in 262 CRC tissues and 41 normal tissues acquired from TCGA RNA-seq datasets. (C) RNF180 expression was assessed via quantitative RT-PCR in 35 pairs of CRC samples collected at our hospital. (D) Representative images of immunohistochemistry (IHC) staining in CRC samples collected at our hospital, showing differential expression of RNF180. Scale bar: 50 μm. (E) Relationship between clinical factors and RNF180 expression in CRC. (F,G) Kaplan-Meier curves of overall survival (OS) (F) and disease-free survival (DFS) (G) of CRC patients based on RNF180 expression. **P < 0.01, ***P < 0.001.

Fig 4: RNF180 interacted with WISP1 and induced K50-linked ubiquitination of WISP1. (A) Purification of RNF180 immunocomplex. Proteins were separated on SDS-PAGE and stained by Coomassie Blue. (B) Immunoprecipitation was carried out with IgG control, anti-RNF180, or anti-WISP1 antibody. The immunoprecipitants were then incubated with the indicated antibodies. (C) Subcellular localization of WISP1 (red) and RNF180 (green) was measured by immunofluorescence. DAPI (blue) was used for nuclear counterstain. Scale bar: 50 μm. (D) RKO cells overexpressing RNF180 were treated with MG132 (10 μM), and the mRNA and protein levels of WISP1 were assessed by quantitative RT-PCR and Western blot. (E) RKO cells transduced with the indicated plasmids were treated with CHX (100 μg/mL), and WISP1 expression was determined by western blot analysis. (F) RNF180-overexpressing RKO cells were immunoprecipitated with WISP1 or IgG antibodies and ubiquitination was evaluated by Western blot. (G) HEK293T cells were co-transfected with either the wild-type Flag-WISP1 (WT) or mutants (K50R, K190R, and K268R), together with myc-RNF180 and His-Ub plasmids, and pull down assay was performed. ***P < 0.001.

Fig 5: RNF180 antagonized WISP1 in regulating tumor growth in mouse. RKO cells stably expressing the indicated genes were subcutaneously injected into nude mice (n = 6 per group). (A) Tumor volume was measured every 3 days for 33 days. (B) At day 33, mice were euthanized, and tumor characteristics were recorded. (C) Representative images of TUNEL staining in xenograft mouse tumors. Apoptotic cells were quantified accordingly. Scale bar: 50 μm. (D) Protein levels of WISP1 and Ki67 in RKO cells that were transduced with the indicated plasmids were measured by Western blot. ***P < 0.001 compared with vector. ###P < 0.001 compared with RNF180.