Figure 4.

KPT-9274 shows specificity for attenuation of PAK4 targets preferentially in RCC cells.

Both RCC cell lines in addition to a normal primary renal proximal tubular epithelial cell line (RPTEC) were grown to 50% and transfected with an siRNA specific to PAK4 or a scrambled sequence control siRNA, then subjected to:

A. immunoblotting and

B. apoptosis assay by flow cytometry.

C. Whole cell lysates were immunoblotted with the indicated antibodies after incubation of the cells with the indicated concentrations of KPT-9274.

D. The subcellular localization of β-catenin in 786-O cells was determined by immunofluorescent staining after 24 hours of treatment with DMSO, 1μM or 5μM KPT-9274. The fluorescence of FITC-conjugated β-catenin (green), TRITC-conjugated phalloidin (F-actin stained: red) and DAPI (nucleus counter-stained: blue) was visualized under a confocal laser-scanning microscope. Scale bar: 20 μm

E. Western blot analysis of β-catenin in the cytosolic and nuclear portions of 786-O cells exposed to DMSO or KPT-9274 for 36 and 72 hours. TBP=TATA-Binding Protein, a nuclear constituent. Please see Materials and Methods for details.