Fig. 2: High matrix stiffness upregulates the expression of USP8 via integrin β1/FAK/YAP axis.

A Western blot and its quantitative results indicating the relative expression of integrin β1, p-FAK, FAK, and YAP protein in DU145/PC-3 cells cultured in different PAAG stiffness. B IHC staining and its quantitative results indicating the different expression of integrin β1, p-FAK, FAK, and YAP protein in PCa tissues of Gleason score < 7, or =7, or >7. C Immunofluorescence detecting the subcellular localization of YAP protein in DU145/PC-3 cells cultured in different PAAG stiffness. D Western blot and relative quantification of p-YAP (S127) and YAP protein. E Venn diagram showing the differentially expressed genes (DEGs) detected by RNA-seq of DU145 cultured in different PAAG stiffness. F RT-qPCR showing the relative transcript level of USP8 in DU145/PC-3 cells cultured in different PAAG stiffness. G Western blot indicating the relative expression of USP8 protein in DU145/PC-3 cells cultured in different PAAG stiffness or in combination with siYAP transfection. H IHC staining and its quantitative results indicating the different expression of USP8 protein in PCa tissues of Gleason score < 7, or, =7, or >7. I Western blot indicating the relative expression of USP8 protein in DU145/PC-3 cells cultured in different PAAG stiffness or in combination with siITGB1 or siFAK transfection. J, K Relative luciferase activity in HEK-293T cells co-transfected with either TEAD2 plasmid or empty vector and either USP8-promoter-WT, or USP8-promoter-MUT vectors. L ChIP-qPCR was used to detect the binding of TEAD2 to the USP8 promoter in DU145 and PC-3 cells using TEAD2 primary antibody, and IgG was applied as a negative control. Data are presented as the mean ± SD of at least three independent experiments and were analyzed with unpaired two-tailed student’s t-test or one-way ANOVA test unless otherwise stated, *P < 0.05, **P < 0.01, ***P < 0.001.