Fig. 5: KCTD9 binding to ZNT9 competes with ZNT9 binding to β-catenin.

A Protein-protein interaction network predicted for KCTD9 using the Search Tool for Retrieval of Interacting Genes/Proteins (STRING) database. B Recombinant Flag-tagged (3xF)-KCTD9 was immunoprecipitated from HCT-116 cells to identify putative KCTD9-binding proteins. Coomassie blue-stained SDS-PAGE gel compares control IgG versus Flag immunoprecipitates with the highlighted bands identified as KCTD9 and ZNT9 using mass spectrometry. Data shown represent three independent experiments. C Venn diagram showing intersecting candidate KCTD9-binding proteins identified from the analyses in A, B. D, E Western blotting verification that ZNT9 co-precipitates with ectopically expressed 3xF-KCTD9 and conversely that KCTD9 co-precipitates with ZNT9 in HCT-116 cells. Data shown represent three independent experiments. F Western blotting analysis of ZNT9, E-cadherin, N-cadherin, vimentin, and SNAIL in LoVo and SW620 cells after knockdown of ZNT9. Data shown represent three independent experiments (top) and relative density were quantified by using ImageJ software (bottom). G Western blotting analysis of β-catenin, c-Myc, cyclin D1, and MMP-7 in total cell lysates and β-catenin in nuclear isolates of the ZNT9 knockdown cells from (F). β-actin and PCNA were used as loading controls for total and nuclear proteins, respectively. Data shown represent three independent experiments (top) and relative density were quantified by using ImageJ software (bottom). H, I SW620 cells were transfected with the indicated constructs and immunoprecipitation (IP) analyses used to detect protein-domain interactions between KCTD9 and the indicated HA-tagged constructs of ZNT9 (FL, P1, P2, and P3) or ZNT9 and the indicated Flag-tagged constructs of KCTD9 (FL, P1, and P2). Data shown represent three independent experiments. J, K Co-immunoprecipitation assays were conducted in SW620 cells measuring interactions between β-catenin and ZNT9 and ZNT9 and β-catenin in the absence or presence of KCTD9 overexpression. Data shown represent three independent experiments (left) and relative density were quantified by using ImageJ software (right). L Mammalian two-hybrid assays in SW620 cells measuring interactions between ZNT9 and β-catenin in the absence or presence of KCTD9 overexpression. F, G are mean ± SD; n = 3 independent experiments, one-way ANOVA with Tukey’s multiple comparison post-test, *p < 0.05, **p < 0.01, ***p < 0.001. J, K are mean ± SD, n = 3 independent experiments, two-tailed Student’s t test, **p < 0.01. L Is mean ± SD, n = 3 independent experiments, two-way ANOVA with Bonferroni’s multiple comparison post-test, **p < 0.01.