Fig 2: DEPDC1B induces EMT in PCa via the Rac1-PAK1 pathway. (A) The levels of N-cadherin, E-cadherin, ß-catenin, snail, slug, and claudin1 protein were detected by Western blotting in DEPDC1B-overexpressing cells combined with NSC23766. (B) The levels of N-cadherin, E-cadherin, ß-catenin, snail, slug, and claudin1 protein were detected by Western blotting in DEPDC1B-knockdown cells combined with Q61L. (C) The levels of N-cadherin, E-cadherin, ß-catenin, snail, slug, and claudin1 protein were detected by Western blotting in DEPDC1B-overexpressing cells combined with Rac1 iRNA. (D) A schematic model of the mechanism underlying the role of DEPDC1B in PCa metastasis and progression

Fig 3: Mutated active Rac1 plasmid (Q61L) recuses the function in DEPDC1B-knockdown cells. (A) The levels of active Rac1, total Rac1, phosphorylated PAK1, and total PAK1 protein were detected by Western blotting in DEPDC1B-knockdown cells combined with Q61L. (B and C) Representative images of cell migration (B) and invasion (C) were analyzed using DEPDC1B-knockdown or control cells combined with Q61L (left panels), and a quantification analysis of the migrated or invaded cell counts (right panels) is shown. (D) Representative images of wound-healing assays using DEPDC1B-knockdown or control cells combined with Q61L in DU145 (left panels) and PC3 (right panels) cells, showing reversing cell motility after using Q61L in DEPDC1B-overexpressing cells. (E) A quantification analysis of cell migration index is shown. (F) Representative images of three-dimensional (3D) cell culture using DEPDC1B-knockdown or control cells combined with Q61L showing rescued cell motility after using Q61L in DEPDC1B-knockdown cells. (G) Cell viability was rescued by using Q61L in DEPDC1B-knockdown or control cells. (H) Colony formation assays were constructed in DEPDC1B-knockdown or control cells combined with NSC23766. (I) A quantification analysis of the colony formation number was shown. Unpaired t-test was used to analyze two groups of data; *P < .05, **P < .01, and ***P<.001. Two-way ANOVA was performed to analyze factorial designed data, # P < .05, ##P < .01, and ###P<.001. Scale bars: 25 µm

Fig 4: DEPDC1B regulates the Rho signaling pathway and binds to Rac1. (A) Representative image of silver-stained SDS-PAGE gels showing separated proteins that were pulled down using Flag-labeled DEPDC1B. Anti-IgG was used as the negative control. (B) The bar graph of top 10 nonredundant enrichment clusters of KEGG using the Metascape website. (C) PPI network visualization in String website showing the proteins that related to DEPDC1B and Rac1. (D) The mass spectrum of a representative peptide fragment of Rac1. (E and F) Western blot analysis determined that DEPDC1B is correlated with Rac1 after performing the pull-down assay with Flag-labeled DEPDC1B (E) and an anti-Rac1 (F) immunoprecipitation antibody. Anti-IgG was used as the negative control protein in the pull-down assay. (G and H) Representative image of the Western blotting analysis of active Rac1, total Rac1, phosphorylated PAK1, total PAK1 protein levels after DEPDC1B-knockdown (G) or -overexpression (H) in DU145 and PC3 cells