Fig 1: WWP1 silencing suppressed proliferation, migration, and invasion of HuCCT1 cells in vitro.A WWP1 protein levels in the four ICC cell lines and normal human intrahepatic biliary epithelial cell (HIBEC) were determined by western blotting. B The efficiency of WWP1 knockdown was estimated by western blotting. C CCK-8 assay, D colony formation assay, E EdU assay were performed to estimate the proliferation of HuCCT1 cells after WWP1 silencing, n = 3. Overexpression of WWP1 again in HuCCT1 cells infected with the lentiviral shWWP1 rescued the ability of proliferation (C, D). F, G Transwell assays were performed to detect the migration (F) and invasion (G) of HuCCT1 cells after WWP1 silencing, n = 3. **P < 0.01, ***P < 0.001.
Fig 2: WWP1 promoted the subcutaneous tumor growth of ICC cells in vivo.A–C Subcutaneous tumor models were applied to explore the impact of WWP1 silencing on proliferation in vivo. A The images of tumors were generated in nude mice using WWP1 silenced HuCCT1 cells, n = 10. B The volumes and weights of subcutaneous tumors between WWP1 silencing and negative control groups. C HE and Ki-67 IHC staining images of subcutaneous tumors. D–F Subcutaneous tumor models were performed to detect the influence of WWP1 overexpression on proliferation in vivo. D The images of tumors were generated in nude mice using WWP1 overexpression RBE cells, n = 7. E The volumes and weights of subcutaneous tumors between WWP1 overexpression and vector groups. F HE and Ki-67 IHC staining images of subcutaneous tumors. **P < 0.01.
Fig 3: WWP1 promoted ICC proliferation and migration via negative regulation of NDFIP1.A HuCCT1 cells were transfected with WWP1 siRNA and NDFIP1 siRNA, then western blotting was used to detect the efficiency of transfection. B–D HuCCT1 cells were transfected with WWP1 siRNA and NDFIP1 siRNA, B EdU assay, C CCK-8 assay, and D colony formation assay were applied to estimate the proliferation of HuCCT1, n = 3. E HuCCT1 cells were transfected with indicated siRNA. Transwell assays were performed to detect the migration of HuCCT1, n = 3. F Images of WWP1 protein levels and NDFIP1protein levels in 4 identical ICC tumors and paired adjacent non-tumor tissues by western blotting assays. G A proposed model of how WWP1 regulates the ICC progression: MYC upregulates WWP1 in ICC cells, thereby targeting NDFIP1 for ubiquitination and degradation to promote ICC cell proliferation, migration, and invasion. **P < 0.01, ***P < 0.001.
Fig 4: MYC transactivated WWP1 gene expression in ICC cells.A The protein level of WWP1 in HuCCT1 cells with MYC knockdown was evaluated by western blotting, n = 3 B The protein level of WWP1 in HuCCT1 cells with SOX-9 knockdown was determined by western blotting, n = 3. C The protein level of WWP1 in HCCC-9810 cells with MYC overexpression was evaluated by western blotting, n = 3. D The mRNA levels of WWP1 in HuCCT1 cells with MYC knockdown and HCCC-9810 cells with MYC overexpression were evaluated by qPCR, n = 3. E The protein level of WWP1 in HuCCT1 cells treated with 10058-F4, a specific c-MYC inhibitor, in the indicated concentrations for 24 h was evaluated by western blotting, n = 3. F The mRNA level of WWP1 in HuCCT1 cells treated with 10058-F4, a specific c-MYC inhibitor, in the indicated concentrations for 24 h were evaluated by qPCR, n = 3. G Schematic representation of MYC responsive elements in the promoter region of WWP1. H Chromatin level of MYC at the promoter region of WWP1 was detected in HuCCT1 cells. Fold enrichment of MYC relative to normal mouse IgG was evaluated by qChIP assay. Fold enrichment of RNA polymerase II at the promoter region of GAPDH served as a positive control. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 5: WWP1 interacted with NDFIP1.A Proteins that interacted with WWP1 were detected by Coomassie blue staining SDS-PAGE gel and mass spectrometry. NDFIP1 was identified as its molecular weight located on one specific band (red arrow). B Gene Ontology (GO) analysis of WWP1-binding proteins identified by mass spectrometry was performed. C WWP1 binding proteins were analyzed by KEGG pathway enrichment. D The protein-protein interaction network among proteins identified by mass spectrometry was constructed by PPI analysis in String Database. E WWP1(red) and NDFIP1 (green) colocalization in indicated cells was performed by confocal microscopy after immunofluorescence staining. F Interaction between WWP1 and NDFIP1 in HuCCT1, HCCC-9810, RBE cells were confirmed by co-IP assays followed by western blotting.
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