Fig. 1.

Characterization of the effect of GAGE proteins on the metastatic potential of breast cancer cells. a Immunohistochemical staining of GAGE proteins in MDA-MB-435-derived cell lines with different metastatic potential as described in Montel et al. [11] (anti-GAGE mAb, clone M4 [9]; DAB). b Western blot analysis of GAGE in CL16 cancer cells transduced with 5 different GAGE-specific lentiviral shRNA vectors (GAGE-shRNA1–5), empty vector (pLKO.1-1 and pLKO.1–2) or untransduced (CL16). c-d GAGE expression (C) and size (D) of primary tumors from CB17 mice implanted with 10⁶ GAGE-shRNA or pLKO.1-transduced CL16 cells and Matrigel (Sigma-Aldrich, St. Luis, Missouri, USA) into the mammary fat pat. e Quantification of the metastasis burden in lungs of mice by staining of the excised and embedded lungs with an antibody specific to human vimentin and scoring using NDP view software. Experimental groups were compared using the Students t-test (p values >0.05 was considered nonsignificant)