Fig. 4

Processing of NY-ESO-1 HLA-Cw3 restricted epitopes following PSMB8 siRNA knockdown in melanoma cell lines. PSMB8 expression was knocked down by siRNA in melanoma cell lines and knockdown was confirmed at mRNA (a) and protein (b) levels following 72 h incubation. Following 72 h of PSMB8 knockdown alongside incubation +/− IFNγ, T-lymphocyte recognition (c–e) or cytotoxicity (f–h) was assessed and compared to cells treated with scrambled control siRNA. (c–e): T-lymphocytes were incubated with melanoma cells for 4 h in presence of Brefeldin A and cytokine secretion was determined by intracellular staining, followed by FACS analysis. f–h: T-lymphocytes were incubated with melanoma cells at a 1:1 effector:target ratio for 24 h at 37 °C, then washed off. T-lymphocyte mediated cytotoxicity was determined by MTS assay and normalized to control wells with no T-lymphocytes. Error bars represent SEM (n = 3). Ns = not significant >0.05; * < 0.05; ** < 0.01; *** < 0.001