Fig. 3

SNX2112 treatment leads to degradation of HSP90 client proteins in PRCC cells (A) PRCC cell lines (UOK345, UOK337, UOK342, UOK332) were treated with different concentrations of SNX2112 (10 nM, 50 nM and 100 nM) as indicated for 24 h, followed by Western Blot analysis. SNX2112 treatment induced the degradation of client proteins such as MET, AKT and inhibits phosphorylated forms of MET (Y1234/1235), AKT (Ser473) and ERK1/2 (Thr202/Tyr204). Three independent experiments were performed, and a representative blot is shown. B Knockdown of AKT1/2 and ERK1/2 proteins confirmed by western blot. PRCC cells with MET activating mutation (UOK345) or copy number gain of WT MET (UOK342) were transfected with siAKT1/2, siERK1/2 or non-targeting control, lysed and subjected to western blot. The percentage knockdown for AKT1/2 or ERK1/2 levels was calculated for cells treated with targeting siRNA for AKT1/2 or ERK1/2 relative to the cells treated with non-targeting control after being normalized with an internal control (β actin). Data are representative of three independent experiments. Bar graphs represent the mean ± SD, n = 3. *p < 0.05 is considered significant and was calculated by the two tailed Student’s t test. C AKT1/2 and ERK1/2 silencing decreases PRCC cell viability. UOK345 and UOK342 cells were transfected with siAKT1/2, siERK1/2 or non-targeting control and effect on cell viability was assessed at 48 h and 72 h post-transfection by Cell-Titer Glo assay. Data are mean ± SD of three independent experiments. *p < 0.05 is considered significant and was calculated by the two tailed Student’s t test. D AKT1/2 and ERK1/2 knockdown leads to diminished 2D-colony formation. Following transfection (48 h) with siAKT1/2, siERK1/2 and non-targeting control siRNA, UOK345 and UOK342 cells were trypsinized and seeded at very low density (1000–1500 cells/well) in six-well plates. Tumor foci were stained with crystal violet (0.5%) after 10–12 days and imaged with microscopy. *p < 0.05 is considered significant and was calculated by the two tailed Student’s t test or other tests