The effects of DBD mutations on PARP-dependent O/N STRESS induced NAD degradation and TRPM2-dependent Ca²⁺ transients.

<p><i>A) PARP-dZF</i> mutant expression levels relative to PARP-WT:<i>Left Panel</i>: 50 µg of cellular protein were loaded into each lane of an 8% SDS-PAGE gel and analyzed by western blotting. Rabbit anti-human PARP-1 polyclonal antibody was used as the primary antibody for immunoblotting of PARP-1 (1∶4000, Alexis Biochemicals), and IR680 conjugated goat anti-rabbit as secondary antibody (1∶3000, Licor Inc). Blots were analyzed on a Licor Odyssey. <i>Middle Panel</i>: Relative transcript abundance of <i>PARP-dZF</i> mutants normalized to <i>PARP-WT</i>, as determined by Q-PCR. <i>Right panel</i>: TRPM2-dependent whole cell currents are similar in <i>PARP-WT</i> and <i>PARP-dZF</i> mutant clones. Average whole cell currents were not statistically different from one another across all cell types. Cells were patched in the whole cell configuration: the pipette solution contained 100 µM mADPR. The I-V relationship and current development across all cell types was characteristic of TRPM2 and identical to that previously shown by our lab <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006339#pone.0006339-Buelow1" target="_blank">[5]</a>. At least 3 whole cell recordings were taken for each cell type. <i>B) Left panel</i>: NAD turnover in <i>PARP-WT and PARP-dZF1</i> DT40 cells. <i>PARP-dZF1</i> did not show NAD degradation over the course of 30 minutes following application of 100 µM MNNG. Stars indicate a p-value of p≤.001 from baseline for all subsequent points. <i>Right panel</i>: TRPM2-dependent Ca²⁺ transients in <i>PARP-WT and PARP-dZF1</i> DT40 cells after stimulation with 500 µM MNNG, as measured by ratiometric analysis of Indo-1 stained cells by FACS. No transients were seen in <i>PARP-KO</i> or <i>PARP-dZF1</i> at 100 µM MNNG (data not shown). <i>C) Left panel</i>: NAD turnover in <i>PARP-WT and PARP-dZF2</i> DT40 cells. <i>PARP-dZF2</i> cells show NAD degradation over the course of 240 minutes following application of 100 µM MNNG. Stars indicate a p-value of p≤.001 from baseline for all subsequent points. <i>Right panel</i>: TRPM2-dependent Ca²⁺ transients in <i>PARP-WT and PARP-dZF2</i> DT40 cells after stimulation with 100 µM MNNG, as measured by ratiometric analysis of Indo-1 stained cells by FACS. No transients were seen in <i>PARP-KO</i> or <i>PARP-dZF2</i> at 100 µM MNNG (data not shown).