Fig. 4: JOSD1 facilitates colon cell progression via Hippo/YAP axis.

A Depletion of JOSD1 reduced the level of YAP protein, which was then restored by overexpressing YAP. HCT116 cells were transfected with either siControl or siJOSD1. They were then subjected to another round of transfection with Flag-YAP or Flag vector after 24 h. Subsequently, the cells were harvested for western blot analysis 48 h later to assess the expression levels of JOSD1 and YAP proteins, with actin serving as the internal control. B Depletion of JOSD1 suppressed the expression of Hippo target genes, which was reversed by YAP overexpression. HCT116 cells were transfected with either siControl or siJOSD1. Subsequently, they underwent a secondary transfection with Flag-YAP or Flag vector after 24 h. RNA extraction from experimental groups was performed using Trizol to assess the impact on the expression of YAP target genes. C Depletion of JOSD1 reduced TEAD luciferase activity in HCT116 cells. YAP overexpression reversed this effect. D The CCK-8 assay measured the cell growth of HCT116 cells. JOSD1 depletion inhibited their ability to proliferate. However, this effect could be reversed by overexpressing YAP. E, F Depletion of JOSD1 reduced the number of colorectal cancer cells that were positive for EdU. YAP overexpression further rescued this effect. Cell proliferation activity was indicated by the absolute cell number. G, H Depleting JOSD1 reduced the migratory ability of colorectal cancer cells. YAP overexpression reversed this effect. I, J Depleting JOSD1 reduced the invasive ability of colorectal cancer cells. YAP overexpression reversed this effect. The mean cell number was calculated and presented with standard deviations. K, L FACs were used to measure the apoptosis of HCT116 cells as indicated. Depletion of JOSD1 promoted apoptosis in HCT116 cells, and YAP overexpression was found to attenuate this effect. The right panel shows a quantitative summary of the apoptosis analysis using FACS. M–O In vivo, YAP overexpression rescued the growth of xenograft tumors from colon cancer cells transfected with siJOSD1. Panels (M), (N), and (O) show the tumor growth curves, weights, and photographs, respectively. P, Q The IHC staining of xenografts revealed the levels of JOSD1, YAP, and Ki67. The quantification of Ki67 positive cells results is shown in the right panel. All data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA.