Functions of the OTU-domain containing proteins in prostate cancer progression. (A) LNCaP-FGC cells transfected with siRNAs targeting different OTU family members were assayed for: (left panel) the knockdown efficiency of the siRNAs measured by quantitative real-time PCR -results are shown as percentage expression of each gene relative to siRNA control transfected cells; (middle panel) cell proliferation and (right panel) matrigel cell invasion. (B) Matrigel invasion assay (upper panel) and western blot analysis of OTUB1 expression (lower panel) in LNCaP-FGC cells transfected with two different siRNAs targeting OTUB1 or a control siRNA, treated with DHT (10 nM). (C) Same as in (B) using LNCaP-FGC cells transfected with either an empty vector or expression plasmids for wild type OTUB1 (OTUB1-WT) or the protease inactive C91S (OTUB1-C91S) variant. (D) Measurements of matrigel invasion and (E) cell proliferation of PC3 cells transfected with either siRNAs targeting OTUB1 or expression vectors bearing OTUB1-WT or the C91S variant, as indicated. In all panels, each column represents the average ± SD of at least four independent replicates and Student´s t test was used for statistical analysis. In (A), *indicates a significant reduction (p < 0.05) of mRNA levels and invasive capacity between LNCaP-FGC cells transfected with control siRNA compared with the different OTUs siRNAs. In (B) and (C), *indicates a statistically significant (p < 0.05) change after DHT treatment in cells transfected with the same siRNA or plasmid. #indicates a significant (p < 0.05) change between cells transfected with siOTUB1, OTUB1-WT or OTUB1-C91S and control transfected cells upon DHT stimulation. In (D), #indicates a significant (p < 0.05) change between cells transfected with siOTUB1, OTUB1-WT or OTUB1-C91S versus control transfected cells.