Figure 5.

NPNT promotes colonization of the lung via its integrin-binding motifs. 66cl4-cells expressing EV, NPNT wild-type, NPNT RGE, or NPNT RGE-AIA mutants were injected into the lateral tail vein to assess tumor cell colonization of the lung using 24-25 mice per group. (A) Lungs were collected after 3 weeks and weighed (mean±SD). **P<.0005 when analyzed by two-tailed t test. The data have been merged from two separate experiments using 14-15 and 9-10 mice per group. (B) RTB assay using genomic DNA from whole lung lysates as template (n=24-25). Graphs show mean RTB ± SD, ***P<.0001 when analyzed by two-tailed t-test. (C) Images of three representative lungs from each group of mice. Macroscopic view of representative lung lobes imaged to detect mCherry positive tumor cells (bottom row). The tissues were imaged using the EVOS FL Auto Cell Imaging System with an inverted microscope and a Sony ICX445 monochrome CCD camera and visualized using 4× objectives. Each image was created using a stitch of several images to cover the entire lung lobe. (D) Representative images of IHC-stained lungs from mice using NPNT antibodies (Atlas Antibodies/Sigma). The top and middle rows show antibody-stained sections at two different magnifications, whereas the lower row shows rabbit IgG isotype staining controls. Scale bars: top and lower row: 100 μm, middle row: 10 μm. Tu: indicates tumor area, Lu: indicates normal lung tissue. Images are representative of a series of samples from five mice.