Supplementary Figure 3 from Nitric Oxide Production Upregulates Wnt/β-Catenin Signaling by Inhibiting Dickkopf-1

PDF file - 136K, iNOS/NO downregulated DKK1 gene expression in cancer cells. A, HCT116 and MCF7 cell lines were treated with NO donor SNAP (500 microM) for 4 hrs. Total RNA was isolated for RT-PCR analysis. B, SW480-DKK1 and SW480-vector (control) cell lines were incubated for 24 hrs and total proteins were isolated for Western blot analysis with the indicated antibodies. C, SW480-DKK1 and SW480-vector cell lines were transfected with pTOP/pFOP for 48 hrs, and the cell lysates were used for luciferase reporter assay. D, SW480-iNOS-DKK1and SW480-iNOS-vector cell lines were incubated for 24 hours. Total proteins were collected for Western blot analysis with iNOS and DKK1 antibodies. E, HCT116 cells were transfected with pCDNA3-hiNOS and pCDNA3-vector, and with pcDNA3.1/V5-His-hDDK1 and pCDNA3.1/V5-His-vector. After 36 hours, total protein was isolated for immunoblotting with c-MYC or Beta-actin antibody.