Skip to main content
. Author manuscript; available in PMC: 2015 Apr 1.
Published in final edited form as: Mol Cancer Res. 2014 Jan 24;12(4):514–526. doi: 10.1158/1541-7786.MCR-13-0505

Figure 6.

Figure 6

Treatment with 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15) inhibited growth of human renal tumor xenografts. The investigations were advanced to a nude mouse xenograft model. A. Experimental time line. SK-NEP-1 cells were injected into the subcapsular space of the left kidney. After two weeks, animals were randomized to receive twice daily intraperitoneal injections of vehicle (n = 7, sterile normal saline, control, 100 μL) or Y15 (n = 8, 30 mg/kg/day, 100 μL). Animals were euthanized after 21 days treatment and their tumors harvested for study. B. Tumor volumes were measured when animals euthanized. Animals treated with Y15 had significantly smaller tumors than animals treated with control vehicle. C. Representative photomicrographs at 40 × of immunohistochemical staining of the formalin fixed, paraffin-embedded SK-NEP-1 xenograft samples. Immunohistochemical staining showed that the tumors from animals treated with Y15 had decreased FAK Y397 phosphorylation (bottom right panel) compared to vehicle treated tumors (top right panel). Negative controls (mouse or rabbit IgG) were performed with each run (inserts top panels). D. To further confirm target knockdown in the tumor specimens, immunoblotting for FAK Y397 was performed on xenograft tumor lysates. Tumors were homogenized and proteins separated on SDS-PAGE gels, immunoblotting for Y397 and total FAK were performed, with a representative immunoblot shown. There was a decrease in FAK phosphorylation (Y397) in the tumors from the animals that received Y15 treatment. These findings were further demonstrated by densitometry, reported as pFAK:FAK ratio. E. Immunoblots from a number of xenograft specimens were analyzed with densitometry. Phosphorylated FAK was expressed as a ratio to total FAK for each blot and normalized to the β-actin for that blot, allowing for a comparison to be made between the saline treated and the Y15 treated tumors. There was a significant decrease in the FAK phosphorylation (Y397) in the tumors from the animals treated with Y15.