Fig. 7: YAP regulates the expression of JOSD1, forming a forward regulatory loop in YAP signaling with JOSD1.

A The JOSD1 genome schematic was analyzed, and the binding region of YAP to the JOSD1 promoter was studied. B The ChIP analysis showed that YAP binds to the JOSD1 promoter region. HCT116 cells were fixed for 30 min and lysed to purify the DNA, using rabbit IgG as a negative control. Primer sequences can be found in the Methods section. The enriched DNA fragments were then analyzed by DNA gel electrophoresis. C, D Cells from HCT116 and SW480 were treated with siRNA to delete YAP, followed by fixation, lysis, DNA enrichment, and ChIP-qPCR analysis. Results indicated a decrease in YAP binding to the JOSD1 gene. E, F Decreased YAP levels result in a reduction of JOSD1 protein. YAP was silenced with siRNA in HCT116 or SW480 cells, followed by cell lysis and protein extraction. Immunoblotting of cell lysates was performed using specified antibodies, with β-Actin serving as an internal control. G, H Depletion of YAP in HCT116 and SW480 cells inhibits JOSD1 mRNA. After transfection of HCT116 and SW480 cells with siControl or siYAP, total RNA was extracted for gene expression analysis. Relative CYR61 and JOSD1 mRNA levels were assessed by qRT–PCR in triplicate for each group. Statistical significance was determined by comparing the expression levels of the target genes. I, J The expression of JOSD1 protein was inhibited in HCT116 and SW480 cells following verteporfin treatment. Proteins were extracted from HCT116 and SW480 cells following treatment with vector and verteporfin, and immunoblotting was conducted with the specified antibodies. K, L Treating HCT116 and SW480 cells with verteporfin led to the inhibition of JOSD1 mRNA. HCT116 and SW480 cells were treated with either vehicle or VP, followed by total RNA extraction after 48 h for gene expression analysis. Each group underwent triplicate testing. M, N Treatment with XMU-MP-1 increased JOSD1 protein expression in HCT116 and SW480 cells. Proteins were extracted from HCT116 and SW480 cells following treatment with vector and XMU-MP-1, and immunoblotting was conducted with the specified antibodies. O, P Treatment with XMU-MP-1 in HCT116 and SW480 cells increased JOSD1 mRNA expression. HCT116 and SW480 cells were treated with either vehicle or XMU-MP-1, followed by total RNA extraction after 48 h for gene expression analysis. Each group underwent triplicate testing. Q–T YAP inhibition reduced JOSD1 expression in the cell membrane of HCT116 and SW480 cells treated with either vehicle or verteporfin. Endogenous JOSD1 was marked in green, and nuclei were stained with DAPI (blue); scale bar, 20 mm. The figure on the right shows the quantitative plots of the fluorescence intensities. All data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA.