Supplementary Figure 1 from FAK Inhibition Abrogates the Malignant Phenotype in Aggressive Pediatric Renal Tumors

Abstract

<p>PDF file - 1930KB, Supplemental Data Figure S1. A., B. PF-573,228 (PF) and 1,2,4,5,-benzenetetraamine tetrahydrochloride (Y15) inhibition of FAK in SK-NEP-1 renal tumor cell lines. Immunoblotting for cleaved caspase 3 was utilized to further demonstrate apoptosis. SK-NEP-1 cells were treated with PF of Y15 for 24 hours and cell lysates collected. Immunoblotting showed increased cleaved caspase 3 following both PF (A) and Y15 (B) treatment in this cell line, indicating apoptosis. C. FAK inhibition with siRNA in G401 human renal tumor cell line. G401 cells were treated with negative control siRNA (siNeg) or siRNA specific for FAK (siFAK, 20, 40, 60 nM) for 24 hours. Immunoblotting for FAK showed decreased FAK expression in the G401 cells beginning with 20 nM siFAK. There was a decrease in the phosphorylation of kinases upstream and downstream from FAK including Jnk, Src and Erk 1/2 following FAK abrogation. Treatment with siFAK did not affect the phosphorylation of Akt kinase. Expression of total Jnk, Src, Akt and Erk 1/2 were not affected by inhibition of total FAK by siRNA. D., E. Combination treatment of PF-573,228 (PF) and 1,2,4,5,-benzenetetraamine tetrahydrochloride (Y15) for inhibition of FAK in human renal tumor cell lines. G401 and SK-NEP-1 cells were treated with PF and Y15 alone or in combination for 24 hours. AlamarBlue assay was used to measure cell viability. Using the method of Chou 26 the combination indices (CI) were calculated and isobolograms constructed. The straight line connects the LD50 of each small molecule when administered alone. CI values (calculated using the LD50 of the molecules when utilized together) falling above this line suggest and antagonistic effect, those below the line a synergistic effect, and those on the line are thought to represent an additive effect. D. In the G401 cell line the CI values were 0.88 and 0.82. E. For the SK-NEP-1 cell line the CI values were 0.98 and 0.94. These values were close to 1 suggesting that the effect of using both PF and Y15 was an additive one.</p>