Fig. 3: High matrix stiffness promotes the progression and immune evasion of PCa via upregulating USP8.

A CCK8 assay showing the cell viability of DU145/PC-3 cells cultured in different PAAG stiffness or in combination with USP8 shRNA transduction. B Representative images and quantitative results of colony formation assay of DU145/PC-3 cells cultured in different PAAG stiffness or in combination with USP8 shRNA transduction. C, D Representative images and quantitative results of Transwell assay of DU145/PC-3 cells cultured in different PAAG stiffness or in combination with USP8 shRNA transduction. E, F The cytotoxic T cell killing ability to DU145/PC-3 cells cultured in different PAAG stiffness or in combination with USP8 shRNA transduction. G–I RM-1 cells, cultured in different PAAG stiffness or in combination with USP8 shRNA transduction, were injected into the flank of mice. Tumor volumes were measured every 3 days. Tumor images, weight and growth curves were obtained at day 18 after dissection. J Flow cytometry was used to quantitatively analyze the proportion of CD3⁺ T lymphocytes in CD45⁺ cells, CD8⁺ T lymphocytes in CD45⁺CD3⁺ cells, and the proportion of GZMB⁺ cells or PRF1⁺ cells in CD8⁺ T lymphocytes in the subcutaneous tumor samples of mice. The X axis represents the different group. K, L USP8^KO DU145/PC-3 cells were constructed and the effect of PAAG stiffness on the cytotoxic T cell killing ability to USP8^KO DU145/PC-3 cells was explored. M, N We transfected USP8 C786A (dead USP8) plasmid into USP8^KO DU145/PC3 cells. The effect of PAAG stiffness on the cytotoxic T cell killing ability to USP8^C786A DU145/PC-3 cells was explored. Data in A and I were analyzed by two-way repeated measures ANOVA test. 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.