Fig. 2: Role of NY-ESO-1 in anoikis and tumor metastasis.

A–D Representative FACS graphs (left) and statistical analysis (right) of cell death in MCF-7 (A), HeLa (B), MDA-MB-231 (C), and SW620 cells (D) that were stably transfected with NY-ESO-1 expressing vectors (NY) or control vectors (CT). E, F Representative FACS graphs (left) and statistical analysis (right) of cell death in A375 (E) and H1299 cells (F) stably transfected with NY-ESO-1-specific shRNAs (shNY-1 or shNY-2) or control shRNAs (shCT), respectively. For (A–F), cells were grown under adherent (Ad) or suspension (Sus) conditions for 48–72 h and harvested for FACS-based cell death analysis using 7-AAD or PI staining kit. **P < 0.01; ***P < 0.001. G Representative photographs (left) and statistical analysis (right) of colonies formed in soft agar by control and NY-ESO-1-stably expressing MCF-7 cells. Scale bar = 500 μm, ***P <0.001. H Representative photographs (left) and statistical analysis (right) of colonies formed in soft agar by control and NY-ESO-1-knockdown A375 cells. Scale bar = 500 μm, ***P < 0.001. I Representative photographs (left) and statistical analysis (right) of metastatic nodules present in the lungs from mice inoculated with A375 cells stably transfected with shCT or shNY-1, respectively. Arrow denotes visible metastatic sites. N = 6 mice per group, ***P < 0.001. J Representative photographs (left) and quantification (right) of metastatic nodules present in the lungs from mice inoculated with indicated MDA-MB-231 cells and treated with vehicle or trametinib (2 mg/kg/d by oral gavage for 5 consecutive days beginning one day after inoculations). The arrows indicate visible metastatic sites. N = 5–6 mice per group, **P < 0.005.