Figure 6. Newly identified unstable pVHL mutants maintain partial pVHL function and are negatively regulated by USP9X.

A. and B. Western blot analysis (A) and infection efficiency (B) of wild-type and mutant pVHL 786-0 cells. 786-0 cells were infected with VHL virus as indicated for 48 hours. Cells were harvested and immunoblotted with indicated antibodies. The infection efficiency of infected 786-0 cells was evaluated by flow cytometry. C. Western blot analysis of wild-type or mutant pVHL protein levels after USP9X reduction. 786-0 cells as indicated were infected with scramble or shUSP9X lentivirus for 48 hours. Protein levels as indicated were evaluated with western blot. D. Western blot analysis of pVHL protein levels in 786-0-HA-I151S cells under CP2005 treatment. 786-0-HA-I151S cells were treated with CP2005 (1.25 μM) for indicated periods of time. E. 786-0-mock and 786-0-HA-I151S cell lines were infected with scramble or shUSP9X lentivirus for 48 hours. 5 × 10² cells as indicated were placed in a 96-well plates and cultured for the indicated number of days. Relative cell numbers were quantified each day. The results represent the mean ± SEM of three independent experiments and were analyzed with two-way ANOVA. *** p < 0.001. F. Relative VEGF mRNA expression levels were determined by qPCR in 786-0-mock and 786-0-HA-L188V cell lines infected with scramble or shUSP9X lentivirus. Expression levels are normalized to the GAPDH mRNA level. The results represent the mean ± SEM of three independent experiments and were analyzed with the Student's t-test. *** p < 0.001; ** p < 0.05.