Figure 1

Pharmacological focal adhesion kinase (FAK) inhibition selectively reduces nucleostemin (NS) levels and impacts anchorage-independent spheroid breast carcinoma growth. (A, B) Growth of dimethyl sulfoxide (DMSO)- or PF-271 (0.1 μM)-treated cells: MCF7, BT474, 468 (MDA-MB-468), 231 (MDA-MB-231), and 4T1L in adherent conditions for 3 days (A) or methylcellulose for 25 days (B). Values are means expressed as percent of DMSO control. (C) Representative lysates of the indicated cells treated with DMSO or PF-271 for 3 days and immunoblotted for pY397 FAK, total FAK, NS (light and dark exposures), B23, and actin. (D) Ratio of NS to actin levels in PF-271-treated and control cells determined by densitometry. (E, F) Immunoblots from tumor lysates (n = 5 independent controls and 5 treated with VS-4718) quantified by densitometry for MDA-MB-231 and 4T1L cells grown orthotopically in mice and treated with 5% sucrose (control) or 0.5 mg/kg VS-4718 [15]. Shown is the ratio of pY397 FAK to total FAK, NS to actin, and B23 to actin. The mean of vehicle-treated tumors was set to 1. (G) Anti-NS immunoblotting of MDA-MB-231 cells with lentiviral shRNA-mediated NS knockdown (NS-1 and NS-2) compared to scrambled (Scr) control. (H, I) Growth of MDA-MB-231 Scr, shRNA NS-1 or NS-2 cells in methylcellulose over 25 days (H) or adherent culture over 5 days (I). For all graphs, values are means (+/− SEM, *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001) of triplicate points from experiments repeated three times.