Skip to main content
Figure 2 | Breast Cancer Research

Figure 2

From: FAK activity protects nucleostemin in facilitating breast cancer spheroid and tumor growth

Figure 2

Genetic focal adhesion kinase (FAK) inhibition impacts MDA-MB-231 spheroid growth and lowers nucleostemin (NS) protein levels. (A) MCF7 cells over-expressing green fluorescent protein (GFP), GFP-FAK-wild-type (WT), or GFP-FAK-kinase-dead (KD) were analyzed for pY397 FAK, total FAK, NS, B23, and actin levels by immunoblotting. (B) MDA-MB-231 cells were transduced with lentiviral scrambled (Scr) or anti-human FAK shRNA. Murine GFP-FAK-WT (FAK-WT) or GFP-FAK-KD (FAK-KD) were stably re-expressed in FAK shRNA MDA-MB-231 cells. Immunoblotting of total FAK, NS, and actin. (C) Densitometry of NS to actin ratios. Values are means ± SD of triplicate points (*P <0.05) from three independent experiments. (D) Histogram of GFP fluorescence as determined by flow cytometry. (E) Adherent growth of FAK-WT and FAK-KD MDA-MB-231 cells. Values are means (± SEM) of triplicate points from two independent experiments. (F) Representative colony growth in methylcellulose (31 days) of FAK-WT and FAK-KD MDA-MB-231 cells. Scale is 0.5 mm. (G) Colony number of FAK-WT and FAK-KD MDA-MB-231 cells. Values are means ± SEM of triplicate points (***P <0.001) from two independent experiments. (H) Immunoblotting for pY397 FAK, total FAK, NS, B23, and actin in FAK-WT and FAK-KD MDA-MB-231 cells. (I) qPCR analyses of NS mRNA levels from FAK-WT and FAK-KD MDA-MB-231 cells. Fold change as compared to actin (± SEM) is shown. Data represent triplicate points from three independent experiments. N.S., no significant difference. (J) NS, B23, and actin immunoblotting analyses from FAK-WT and FAK-KD MDA-MB-231 cells cultured in methylcellulose for 3 days, and treated or not 3 hours prior to cell lysis (as indicated) with MG132 proteasome inhibitor (40 μM).

Back to article page