Figure 5.

Release of Mitochondrial NAD⁺ Antagonizes PARP Inhibitor Treatment

(A) U2OS cells were treated with PJ34, olaparib, talazoparib, or niraparib, and mitochondrial ADP-ribosylation was subsequently assessed via IF using the anti-ADPr antibody.

(B) U2OS ARTD1^−/− cells were treated with either rotenone or H₂O₂, and the mitochondrial and nuclear ADP-ribosylation were assessed via IF with our anti-ADPr antibody.

(C) U2OS cells were co-treated with MMS and olaparib, rotenone or a combination for 2 h and after pre-extraction, chromatin-bound ARTD1 was analyzed via IF.

(D) U2OS cells were pre-treated with PJ34 or olaparib, then subjected to an FCCP pulse followed by H₂O₂ and analyzed via IF using an anti-PAR antibody. ^∗ = 5-fold increased concentration of inhibitors. Representative pictures are shown in the upper panel; the quantification of the nuclear PAR signal is shown in the lower panel. The quantifications of all signals (mitochondrial and nuclear) were normalized as described in Figure 1, and the y axes of all plots are depicted as log10 scale. Scale bars indicate 20 μm.