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. Author manuscript; available in PMC: 2016 Sep 15.
Published in final edited form as: Oncogene. 2013 Jan 21;33(4):461–473. doi: 10.1038/onc.2012.608

Figure 8. Nodal knockdown reduces spontaneous metastasis to the liver in NOD/SCID/IL-2γ receptor null mice.

Figure 8

500,000 231+ishC or 231+ishN cells were injected into the right thoracic mammary fat pad of NSG mice. Doxycyclin was administered 1 week following injection. Mice were sacrificed when tumors reached 1.0–1.5 cm in diameter and lung and livers were analysed macrosopically or using H&E stained sections. (A) Western blot analysis demonstrating that MDA-MB-231 cells transfected with a Nodal-targeted doxycyclin (Dox)-inducible shRNA (231+ishN) express lower levels of Nodal protein upon administration of Dox compared to 231+ishN cells −Dox, and compared to MDA-MB-231 cells transfected with a scrambled control Dox-inducible shRNA (231+ishC) +/− Dox. (B) Immunohistochemical staining (brown) of Nodal in 231+ishC and 231+ishN derived tumors confirming that Nodal was knocked down in 231+ishN + Dox tumors as compared to all other groups. Bars equal 50 μm. (C) Images of lung sections stained with H&E across treatment groups. Bar equals 1 mm. (D) Quantification of tumor burden in lungs show no significant differences between treatment groups (n=10). Each point represents the average tumor burden for one animal and the filled diamond indicates the median value. (E) Bright field images of livers (top panel) or of liver sections stained with H&E (bottom panel) taken from mice injected with 231+ishC or 231+ishN cells and fed either normal or Dox-containing chow. Metastases have been outlined by a white dashed line and bar equals 1 mm. (F) Quantification of tumor burden in livers show a significant decrease in metastasis in 231+ishN +Dox mice compared to all other groups (n=10, p<0.05). Each point represents the average tumor burden for one animal and the filled diamond indicates the median value. For all graphs, different letters indicate a significant difference. (B) Immunohistochemical staining (brown) of Nodal in primary tumor, lung and liver lesions from mouse injected with 231+ishN and treated with Dox. Note that Nodal expression is regained in the liver lesion. Bars equal 50 μm.