Fig. 1.

PAR polymer induces neuronal cell death. (A) Representative fluorescent microscopic images of PI (red) and Hoechst (blue) staining after treatment of neurons with BioPorter-mediated delivery of PAR polymer, PAR polymer + PARG, PAR polymer + PD1, and poly(A). (B) Silver staining of purified PAR polymer, showing degradation of PAR polymer after incubation with PARG (PAR+PARG) and PD1 (PAR+PD1). These experiments (A and B) have been replicated in separate experiments at least three times with similar results. (C) Quantitative analysis of Hoechst- and PI-stained WT neurons after BioPorter-mediated delivery of PAR polymer, PAR polymer + PARG, PAR polymer + PD1, and poly(A). Purified PAR, cleaved PAR by either PARG or PD1 pretreatment, or synthesized poly(A) was diluted with PBS, mixed with BioPorter reagent, and added to neuronal cultures at a final concentration of 80 nM in serum-free MEM. The neurons were cultured for 3 h to allow the polymers to be imported into neurons and then subjected to fluorescent microscopy and quantitative computer-assisted cell counting after Hoechst and PI double staining 24 h later. Data are the mean ± SD, n = 3; ∗, P < 0.05. (D) PAR polymer can also induce cell death in PARP-1 KO neurons. PAR polymer (80 nM) and cleaved PAR (80 nM PAR polymer + PARG) was delivered into neurons by BioPorter-mediated delivery, and cell death was analyzed by Hoechst and PI staining. Data are the mean ± SD, n = 3; ∗, P < 0.05.