RhoA activity mediates the effects of OTUB1 on p53 expression and matrigel invasion in PCa cells. (A) Western blot analysis for the indicated proteins and RhoA activity assay (left panel) of LNCaP-FGC cells transfected with p53 siRNA or control siRNA and treated with or without DHT. Matrigel invasion assay for the same cells is shown on the right. Quantification of p53 expression relative to beta-Actin levels is indicated. p53 levels in untreated-control transfected cells were set as 1. Western blot and matrigel invasion assays as in (A) were performed using cells transfected with RhoA shRNA or control plasmid (B), wild type (WT), constitutively active (Q63L) or dominant negative (DN) alleles of RhoA (C) and the combination of WT-RhoA and siRNA against OTUB1 (E) in the presence of DHT or control vehicle. (D) RhoA activity assay as in (A) on PC3 cells transfected with siRNAs targeting OTUB1 or control siRNA. In invasion assays, each column represents the average ± SD of at least four independence replicates. *indicates statistically significant (p < 0.05) changes after DHT treatment in cells transfected with the same siRNA or plasmid. #indicates significant (p < 0.05) differences between control transfected cells versus cells tranfected with other siRNAs or plasmids in the presence of DHT. & indicates significant (p < 0.05) differences between cells transfected with siOTUB1 and cells transfected with siOTUB1 and WT-RhoA in the presence of DHT. αindicates significant (p < 0.05) differences between cells transfected with Q63L-RhoA and control cells in the absence of DHT.