Fig. 6: KCTD9 regulates the stability of β-catenin.

A Western blot analysis comparing the expression of β-catenin in SW620 cells without (pCDH) or with overexpression of KCTD9 (pCDH-KCTD9) in untreated or MG132 treated cells (10 μM MG132 for 12 h). β-actin was used as a loading control throughout. Data shown represent three independent experiments (bottom) and relative density were quantified by using ImageJ software (top). B, C Western blot comparing the expression of β-catenin in SW620 cells without (pCDH) or with overexpression of KCTD9 (pCDH-KCTD9) after treatment with cycloheximide (50 μg/ml) for the indicated times. Accompanying densitometric quantitation of β-catenin relative to β-actin (C). D, E Western blot comparing the expression of β-catenin in SW620 cells without (sh-ctrl) or with knockdown of ZNT9 (shZNT9) after treatment with cycloheximide (50 μg/ml) for the indicated times. Accompanying densitometric quantitation of β-catenin relative to β-actin (E). D Represent three independent experiments. F SW620 cells were infected with the indicated combinations of sh-ctrl, sh-ZNT9, pCDH, or pCDH-KCTD9 lentiviruses, followed by co-transfection with HA-ubiquitin. Cells were then pretreated with MG132 (10 μM) for 12 h before immunoprecipitating β-catenin with polyubiquitination detected using anti-HA antibodies. Data shown represent three independent experiments. G Western blot comparing the levels of KCTD9, β-catenin, E-cadherin, N-Cadherin, SNAIL, vimentin, c-Myc, cyclin D1, and MMP-7 in HCT-116 cells infected with the indicated combinations of sh-ctrl, sh-KCTD9, and sh-β-catenin lentiviruses. Data shown represent three independent experiments (left) and relative density were quantified by using ImageJ software (right). H qPCR analysis of samples from G comparing the mRNA levels of c-Myc, cyclin D1, and MMP-7. I Assessment of cell viability in the cells from G measured using CCK-8 assays. J Assessment of proliferation in the cells from G measured using Count Star (Countstar BioTech) over 1–4 days by cell counting. K–M Assessment of migration and invasion in the cells from G using Transwell™ assays. Representative images of migrating/invading cells after 24 h (scale bar=200 µm) K with comparative quantitation of total migrating (L) or invading (M) cells. K Represent three independent experiments. A, G, I, L, M are mean ± SD; n = 3 independent experiments, one-way ANOVA with Tukey’s multiple comparison post-test, ns, not significant, ^*p < 0.05, **p < 0.01, ***p < 0.001. C, E, H, J are mean ± SD, n = 3 independent experiments, two-way ANOVA with Bonferroni’s multiple comparison post-test, ns not significant, **p < 0.01, ***p < 0.001.