USP9x levels correlate with poor outcome in ccRCC. (a) Analysis of USP9x mRNA level comparing normal control tissue and tumor samples from the indicated TCGA data sets. Gene expression values were normalized to a fixed upper quartile value of 1 000 using RNA-Seq by Expectation maximization and represented as scatter plots. Statistical significance was determined using Mann–Whitney tests. (b) Plot of disease-free survival for ccRCC patients as a function of USP9x transcript level. Blue denotes patients with bottom quartile mRNA expression; red denotes patients with top quartile expression and black denotes the middle 50% of patients. A new tumor diagnostic indicator or death was scored as events. Cox proportional hazards regression model was used to calculate P-values. (c) Plot of the histopathology scores for USP9x protein staining of matched normal and tumor samples. Individual tissue spots were examined by an experienced pathologist to confirm the tissue spot identity and visually assigned a score of − (no staining),+ (weak staining of <10% of tissue), ++ (weak staining of 10–25% of tissue), +++ (weak to moderate staining of up to 50% tissue), ++++ (moderate to strong staining of 50–75% of tissue), +++++ (moderate to strong staining of >75% of tissue). In the majority of normal samples and tumors, USP9x staining was localized near the cell membrane, with little or no cytoplasmic staining. However, a small number of tumors showed predominantly cytoplasmic USPx. ccRCC, renal clear cell carcinoma; TCGA, the Cancer Genome atlas.