Figure 3

Focal adhesion kinase (FAK)-kinase-dead (KD) MDA-MB-231 tumors are small with low nucleostemin (NS) levels. (A) Mean volume of FAK-WT (black, n = 8) and FAK-KD (gray, n = 9) MDA-MB-231 cells grown as orthotopic breast tumors in NOD/SCID mice from day 33 to 57 (± SEM, *P <0.05, **P <0.01; ***P <0.001). (B) Final FAK-WT or FAK-KD tumor mass at day 57. Values are means (± SEM, ***P <0.001). (C) Representative images of FAK-WT and FAK-KD tumors at day 57 (scale 0.5 cm.) (D) Mean number of spontaneous breast to lung metastases as visualized by fluorescence (+/− SEM, * P <0.05). (E) Protein lysates from FAK-WT (n = 7) or FAK-KD (n = 7) MDA-MB-231 tumors analyzed by immunoblotting for pY397 FAK, total FAK, nucleolin, NS, B23, and actin. (F) Immunoblots in panel E were quantified by densitometry. Values (from seven tumors) are means (± SEM) and displayed as the ratio of FAK pY397 to total FAK, nucleolin to actin, NS to actin, and B23 to actin. FAK-WT values for each comparison were set to 1 (*P <0.05, **P <0.01). (G) Representative anti-NS immunohistochemical staining (diaminobenzidine, DAB) of FAK-WT and FAK-KD tumors. Tumors were counterstained with methyl green (scale 100 μm). (H) FAK immunoprecipitates (IPs) from three FAK-WT and three FAK-KD tumors were analyzed for associated B23 nucleolar protein by immunoblotting.