Additional file 4.

A TIFF file containing a figure showing that luminescent, metastatic 4T1 cells retain FAK knockdown in vivo, and that FAK knockdown has no effect on pulmonary invasion with tail injection. (a) Luminescent imaging of serially diluted 4T1 cells initially engineered to express firefly luciferase and subsequently scrambled (i.e., scram) or FAK-specific (shFAK) shRNAs. Data show that both 4T1 cell lines expressed identical quantities of luciferase. (b) Immunoblotting control (i.e., scram) and FAK-deficient (shFAK) 4T1 cells before their inoculation (pre-engraftment) into the mammary fat pads of Balb/C mice showed that FAK deficiency was maintained in metastatic 4T1 lesions. Subsequent to isolation from lungs of tumor-bearing mice, cells were cultured in the presence of Zeocin (antibiotic selection of firefly luciferase) (Metastatic) and similarly assayed for FAK expression. Immunoblotting for β-actin was performed as a loading control. (c) Control (i.e., scram) and FAK-depleted (shFAK) 4T1 cells expressing firefly luciferase were injected into the lateral tail vein of Balb/C mice (1 × 10⁵ cells/mouse), and pulmonary invasion was measured at the indicated time points after injection. Data are expressed as the mean (± SEM; n = 10) area flux measurements normalized to the initial pulmonary flux measurement taken upon injection (T0) to correct for injection efficiency.

Format: TIFF Size: 3.8MB Download file

Wendt and Schiemann Breast Cancer Research 2009 11:R68   doi:10.1186/bcr2360