GzmA inhibition of PARylation within cells is partially rescued by expressing K498A PARP-1. PARP-1^−/− MEF cells, transiently transfected to express either WT or K498A PARP-1, were treated with MNNG or PFN and/or GzmA for 20 or 60 minutes. Flow cytometry in cells stained with PAR antibody measured PARP activation in response to DNA damage. (A) Immunoblot shows comparable expression of WT and K498A PARP-1 in PARP-1^−/− MEFs using antibody to C-terminal PARP-1. (B) Shows representative flow cytometry plots, and (C) the mean and SD of the percentage of PAR⁺ cells in 3 independent experiments. Neither PFN nor GzmA alone triggers PAR synthesis. PARP-1 activation to alkylating DNA damage is equivalent in cells expressing WT and K498A PARP-1. However, PARP-1 activity in response to DNA damage by GzmA (A) and PFN (P) is significantly enhanced by mutating the GzmA-favored cleavage site. The gray symbols refer to control cells just treated with PFN (P).