Fig. 5: Identification of AMDHD1 downstream effectors in TGF-β pathway.

A Coomassie blue staining showing the results of an immunoprecipitation assay with AMDHD1. B Flow diagram of the bioinformatic analyses and selection of downstream gene candidates targeted by AMDHD1. Venn diagram depicts the overlap between 483 differentially expressed proteins upon AMDHD1 over-expression (yellow) and 614 AMDHD1-bound proteins (blue). Among 17 overlapping proteins, 14 proteins were up-regulated and 3 proteins were down-regulated in HUCCT1/AMDHD1-OV cells. 2 downstream candidate proteins (SMAD4 and SMAD2) were finally selected through STRING functional enrichment analyses. C Western blot was used to detect the SMAD4 levels in AMDHD1 over-expressed and knockdown cells. D Double-label immunofluorescence staining for the intracellular localization of AMDHD1 and SMAD4 in HUCCT1 cells. HEK293T cells were transfected with Flag-tagged AMDHD1 and MYC-tagged SMAD4 expression plasmids as indicated, whole cell lysates were extracted and immunoprecipitated with anti-Flag antibodies (E) or anti-MYC antibodies (F). G Lysates of HUCCT1 cells were immunoprecipitated with SMAD4 antibody or normal rabbit IgG, then subjected to immunoblotting with AMDHD1 or SMAD4 antibody. H The schematic illustration of SMAD4 and its truncated fragments were shown. I Total lysates from HUCCT1 cells overexpressing MYC-tagged full-length or truncated SMAD4 were subjected to IP with anti-MYC Ab, followed by western blotting using the indicated antibodies (Abs). Ig heavy chain (H chain) was used as a loading control. J, K HUCCT1/AMDHD1-OV cells were transfected with SMAD4-shRNA and the protein levels of AMDHD1, p21, PAI-1, c-Myc, E-cadherin, N-cadherin and Snail were analyzed by immunoblotting. CCK-8 assays (L), colony formation assays (M) and transwell assays (N) were performed in HUCCT1/AMDHD1-OV cells transfected with SMAD4-shRNA. All ns not significant, ***P < 0.001. Scale bars: 200 μm. P values were assessed using two-tailed t-tests and ANOVA followed by Dunnett’s tests for multiple comparison in (B, L, M, N).