Fig 1: Overexpression of DPT inhibits Wnt/ß-catenin signaling. (A) ß-catenin expression in the nucleus in response to DPT-overexpression. (B) Immunofluorescence analysis of ß-catenin levels in DPT-overexpressing HCC cells. Cells were fixed in 4% PFA, treated with pepsin, and dehydrated using a gradient ethanol series. Cells were permeabilized in Triton X-100, blocked in PBS plus goat serum, and probed with anti-ß catenin antibodies and the appropriate secondary antibodies. Cell nuclei were counter-stained with DAPI. Representative confocal images were shown. (C) Expression of Wnt-related genes. Black line: fold-change in gene expression of 1. Pink line: desired fold change in gene expression threshold. (D) Expression of CXXC4, ß-catenin, Cyclin D1 and P27 assessed by Western blotting. Data are the mean ± SD and were compared using a student's t-test. *P < 0.05.
Fig 2: DPT levels in HCC tissue. (A) IHC analysis. (B) Representative Western blotting and (C) qPCR analysis of DPT levels in HCC vs. non-cancerous tissue. (D) Analysis of overall survival of patients with different DPT expression. All data are shown relative to GAPDH expression and were compared using a Student's t test.
Fig 3: Exogenous DPT expression inhibits the CSC phenotypes of HCC. (A) Representative images of the tumor spheres in response to DPT overexpression. (B) Western blotting analysis. (C) Huh7 and HCCLM3 cells overexpressing DPT were transfected with TOP/FOPFlash reporter plasmids. Luciferase activity was assessed after 24 h of transfection. Data are the mean ± SD of triplicate experiments. Data were compared through a Student's t test. **P < 0.01.
Fig 4: CXXC4 inactivates Wnt through CXXC4. (A) CXXC4 and ß-catenin levels in shDPT, shCXXC4 or shDPT cells assessed through Western blotting analysis. (B) Effect of shDPT, shCXXC4 or shDPT and CXXC4 on colony formation. (C) Huh7 cells were treated with shDPT, shCXXC4 or shDPT and CXXC4 for 24 h and stained with EdU and DAPI. (D) Proposed model: Indicating that DPT upregulates the expression of CXXC4. CXXC4 could stabilize the degradation of ß-catenin and reduce ß-catenin expression.
Fig 5: DPT inhibits HCC cell growth and tumorigenicity. (A) DPT levels in the indicated HCC cell lines. (B) DPT overexpression assessed by Western blotting. (C) Cell growth and MTT assays in response to exogenous DPT overexpression. (D) HCC cell lines were treated with DPT for 48 h, EdU treated and fixed. The incorporation of EdU was assayed using a BeyoClick Kit. Representative images are shown. (E-G) Tumor growth curves and weights in response to exogenous DPT expression. Data were analyzed using a Student's t test. Data are the mean ± SD. **P < 0.01.
Supplier Page from Abcam for Anti-DPT/TRAMP antibody [EPR22845-83]