Figure 6
Figure 6. GzmA cleavage of PARP-1 enhances apoptosis and reduces necrosis of target cells. PARP-1−/− MEFs, transiently transfected with expression plasmids encoding WT or K498A PARP-1 (A-C) or PARP-1 fragments (D-E), were treated with GzmA and PFN and assayed for cell death by fluorescein isothiocyanate-annexin V and PI staining or ATP content. In (A) and (E), mean and SD of the proportion of annexin V and/or PI+ cells from 3 independent experiments are shown. (B) Representative annexin V and PI dot plots (top) and the mean and SD of 3 experiments (bottom) 4 hours after treating cells overexpressing WT (■) or K498A (□) PARP-1 with PFN and 2 μM GzmA. Expression of GzmA-resistant K498A PARP-1 increased the percentage of viable annexin V−PI− cells, but the dead cells were more likely to die of necrosis (annexin V−PI+) than apoptosis (annexin V+). The differences for each subgroup of annexin V and PI staining between WT and K498A PARP-1–expressing cells were all significant (P < .01). (C) GzmA treatment depletes cellular ATP in a PARP-1–dependent manner. ATP was measured 1 hour after treatment with nothing (gradient bar), PFN alone (patterned bar), GzmA alone (white), or both (increasing concentrations of Gzm indicated by color change from gray to black). ATP depletion requires PARP-1 because ATP levels were unchanged in PARP-1−/− MEFs. Cleavage of PARP-1 reduces ATP depletion because cells expressing GzmA-resistant K498A PARP-1 are more depleted of ATP than cells overexpressing WT PARP-1. Data shown are the mean and SD of 3 independent experiments (*P < .01; **P < .005). (D) Shows the expression of PARP-1 fragments by immunoblot probed with a mixture of antisera that recognize N-terminal and C-terminal PARP-1. (E) Cells expressing N-terminal PARP-1 had increased susceptibility to GzmA. Cells were transfected to overexpress WT (♦) or K498A (■) PARP-1 (A) or PARP-11-498 (▴) or PARP-1499-1014 (•) or with vector (×). Cell death of control cells exposed to GzmA, but not PFN, is indicated by the corresponding open symbols and dashed lines.

GzmA cleavage of PARP-1 enhances apoptosis and reduces necrosis of target cells. PARP-1−/− MEFs, transiently transfected with expression plasmids encoding WT or K498A PARP-1 (A-C) or PARP-1 fragments (D-E), were treated with GzmA and PFN and assayed for cell death by fluorescein isothiocyanate-annexin V and PI staining or ATP content. In (A) and (E), mean and SD of the proportion of annexin V and/or PI+ cells from 3 independent experiments are shown. (B) Representative annexin V and PI dot plots (top) and the mean and SD of 3 experiments (bottom) 4 hours after treating cells overexpressing WT (■) or K498A (□) PARP-1 with PFN and 2 μM GzmA. Expression of GzmA-resistant K498A PARP-1 increased the percentage of viable annexin V−PI− cells, but the dead cells were more likely to die of necrosis (annexin V−PI+) than apoptosis (annexin V+). The differences for each subgroup of annexin V and PI staining between WT and K498A PARP-1–expressing cells were all significant (P < .01). (C) GzmA treatment depletes cellular ATP in a PARP-1–dependent manner. ATP was measured 1 hour after treatment with nothing (gradient bar), PFN alone (patterned bar), GzmA alone (white), or both (increasing concentrations of Gzm indicated by color change from gray to black). ATP depletion requires PARP-1 because ATP levels were unchanged in PARP-1−/− MEFs. Cleavage of PARP-1 reduces ATP depletion because cells expressing GzmA-resistant K498A PARP-1 are more depleted of ATP than cells overexpressing WT PARP-1. Data shown are the mean and SD of 3 independent experiments (*P < .01; **P < .005). (D) Shows the expression of PARP-1 fragments by immunoblot probed with a mixture of antisera that recognize N-terminal and C-terminal PARP-1. (E) Cells expressing N-terminal PARP-1 had increased susceptibility to GzmA. Cells were transfected to overexpress WT (♦) or K498A (■) PARP-1 (A) or PARP-11-498 (▴) or PARP-1499-1014 (•) or with vector (×). Cell death of control cells exposed to GzmA, but not PFN, is indicated by the corresponding open symbols and dashed lines.

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