Fig 7. Single KO of GrzmB does not affect YT cell killing of PAO1.

(A) PAO1 (1000 CFU/well) incubated alone or in the presence of pNK cells treated with various concentrations of the granzyme B inhibitor ZAAD-CMK or DMSO (control). (B) Untreated pNK treated with 50μM ZAAD-CMK, 0.25μM granzyme A inhibitor (Futhan) or both and cultured for 6 h then plated to determine CFU. * = P≤0.05 and ** = P≤0.01. (C) Western blot analysis of Grnz B in YT cells (1x10⁶ cells), PAO1 or culture media from 6 h co-cultures at a 10:1 ET ratio. PAO1 was separated from YT cells and culture media using a series of centrifugation and washes with sterile distilled water to separate the bacteria which were lysed with 2x Sample Buffer and boiled. Bands were revealed using (1:1000) mouse antibodies specific for human Grzm B. Rabbit antibody specific for human beta actin and a rabbit antibody specific was E. coli RNA polymerase were used as loading controls for the YT and PAO1 cells respectively. (D) Western blot analysis of wild type and granzyme B knockout (C8, D8 and E9) YT cells (5x10⁵ cells). Blotted using (1:1000) mouse antibodies specific for human Granzyme B (clone: m3304b06). (E) PAO1 incubated alone or in the presence of wild type or GrzmB knockout YT cells and cultured for 6 h before determining CFU. (F) PAO1 incubated alone or in the presence of WT or GrzmB KO YT cells for 6 h. Nitrocefin (1mM) was added for 12 h. 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.