Fig. 4: KCTD9 inhibits the EMT and Wnt signaling in CRC cells.

A, B Western blotting analysis of the key EMT markers E-cadherin, N-cadherin, vimentin, and SNAIL in LoVo and SW620 cells after overexpression of KCTD9 or after knockdown of KCTD9 in HCT-116 and DLD-1 cells. β-actin was used as a loading control throughout. Data shown represent three independent experiments (top) and relative density were quantified by using ImageJ software (bottom). C, D Pathway enrichment analysis using the enrichR package. The TCGA-COAD dataset was reanalyzed based on low versus high KCTD9 expression and the 169 DEGs that were upregulated in low KCTD9 expressing CRC tumors used to interrogate four pathway enrichment annotation databases (KEGG_2021_Human, Panther_2016, MSigDB_Hallmark_2020, and WikiPathway_2021_Human). E, F Western blotting analysis of β-catenin, c-Myc, cyclin D1, and MMP-7 in total cell lysates and β-catenin in nuclear isolates after overexpression of KCTD9 in LoVo and SW620, or after knockdown of KCTD9 in HCT-116 and DLD-1 cells . PCNA was used as a loading control for nuclear proteins. Data shown represent three independent experiments (top) and relative density were quantified by using ImageJ software (bottom). G, H Control or KCTD9-knockdown HCT-116 cells were subjected to immunofluorescence staining against β-catenin. Representative confocal images show β-catenin staining alone (green) or in combination with nuclear staining using DAPI (blue) (G). The percentage of cells with coincident staining of β-catenin in the nucleus was quantitated (H). G Represent three independent experiments. I TOP Flash/FOP Flash luciferase reporter assays were conducted in the indicated control or KCTD9-knockdown HCT-116 cells to measure TCF/LEF transcriptional activity. A, E are mean ± SD, n = 3 independent experiments, two-tailed Student’s t test, ^*p < 0.05, **p < 0.01, ***p < 0.001. B, F, H 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. I Is mean ± SD, n = 3 independent experiments, two-way ANOVA with Bonferroni’s multiple comparison post-test, ***p < 0.001.