PMC

Expression of K-RAS4A in the RCC cell lines. Electrophoresis shows the presence of the 100 bp amplified K-RAS4A in three samples from RCC4 cells.

Effect of spironolactone treatment on detection of K-RAS protein by Western blotting. Addition of spironolactone to cultured RCC4 cells results in reduced expression of K-RAS compared to basal culture conditions.

Staining (a) for the mineralocorticoid receptor seen in the nuclei of clear cell RCC and (b) for its gatekeeper enzyme 11β-hydroxysteroid dehydrogenase-2 on the cell membrane and peripheral cytoplasm in these immunoperoxidase preparations (IHC × 20 original objective magnification).

Expression of K-RAS4A in renal cell carcinoma. 1.5% agarose gel showing the presence of K-RAS4A in two examples of clear cell renal carcinomas, and the size of the PCR product was approximately 100 bp indicating K-RAS4A is expressed in renal carcinomas.

Western blot analysis demonstrates that following treatment of both renal carcinoma cell lines with si RNA specific for K-RAS4A, there is knock-down of K-RAS protein. There was also a marked reduction in the phosphorylated forms of Raf, Akt and S6 riboprotein.

Effect on cell population by spironolactone treatment. Treatment of RCC4 plus vector cell cultures led to a 6.5-fold reduction in cell number over 72 h when compared to basal culture conditions. A similar 6.3-fold reduction was seen in RCC4 plus VHL cells (Data not shown).