Fig. 3: NY-ESO-1 protects cancer cells from anoikis by promoting ERK1/2 activation.

A GSEA for enrichment of genes involved in MAPK (left) and ERK1/2 pathway (right) in NY-ESO-1 stably transfected MCF-7 cells (NY) versus control cells (CT). B, C Immunoblot analysis of indicated proteins in tumor cells with or without NY-ESO-1 overexpression or knockdown. Blots of A375 cells in (B, C) were exposed for different times to better show the effects of NY-ESO-1 overexpression or knockdown. D Immunoblot analysis of ERK1/2 activation (p-ERK1/2) in tumor cells stably expressing NY-ESO-1 and control cells grown in adherent (Ad) or suspension (Sus) conditions. E Immunoblot analysis of ERK1/2 activation in A375 cells with stable NY-ESO-1 knockdown and control cells cultured under adherent (Ad) or suspension (Sus) conditions. For panels (B–E), the relative intensity of pERK1/2 normalized to loading controls is shown. The pE/G ratio denotes pERK1/2 normalized to GAPDH, and the pE/T ratio represents pERK1/2 normalized to β-Tubulin. F–H Representative FACS graphs (left) and statistical analysis (right) of cell death in MCF-7 (F), MDA-MB-231 (G), and SW620 cells (H) that were stably transfected with NY-ESO-1 expressing vectors (NY) or control vectors (CT) when they were grown in suspension conditions with or without PD98059 (50 μM) or trametinib (1 μM). I, J Representative FACS graphs (left) and statistical analysis (right) of cell death in A375 (I) and H1299 (J) cells that were stably transfected with NY-ESO-1-specific or control shRNAs when they were grown in suspension conditions with or without PD98059 (50 μM) or trametinib (2 μM).