Fig 4. Perforin and granulysin are not required in YT cell killing of PAO1.

pNK (A) and YT (B) cells were treated with concanamycin A (CMA) or control for 2 h to inhibit the perforin-mediated cytotoxic pathway. PAO1 (1000 CFU/well) was added for 6 h. CFU counts were used to determine number of viable PAO1. Western blot of wild type or Prf KO YT cells (5x10⁵ cells) (C) or wild type or GRNL KO YT cells (5x10⁵ cells) (D) were blotted using (1:1000) mouse antibodies specific for human perforin (Clone: δG9) or human granulysin (clone: F-9). Rabbit antibody specific for human beta actin was used as a loading control. (E) YT cell cytotoxicity against 721.221 tumour target cells. 722.221 cells were stained with CFSE prior to the assay to distinguish them from effector cells. The membrane permeability dye 7AAD was used to assess tumour cell death. Percent killing was determined by the percentage of red and green fluorescent cells divided by the total green fluorescent population. Conditions were carried out in n = 3 wells (mean ± SEM) and the graph is representative of n ≥ 2 biological replicates. (F) PAO1 incubated alone or in the presence of wild type or perforin knockout YT cells and cultured for 6 h, then plated for CFU counts. (G) PAO1 (1000 CFU/well) incubated alone or in the presence of wild type or granulysin knockout YT cells and cultured for 6 h, then plated to determine CFU. In all co-culture experiments, conditions were carried out in n = 4 wells (mean ± SEM) and the graph is representative of n ≥ 3 biological replicates. * = P≤0.05, ** = P≤0.01, *** = P≤0.001.