2.3. PARP Activity Assay

A GCLP-validated assay was used to measure DNA damage-activated PARP activity in permeabilised cells in the presence of an NAD+ substrate (350 nM) and a 12 mer palindromic double-stranded oligonucleotide (10 µg/mL) (Invitrogen, Waltham, MA, USA) to activate PARP1 via immunological detection of the product (PAR) using 10H Ab (Enzo life sciences, Farmingdale, NY, USA) and secondary HRP-conjugated goat anti-mouse Ab (Dako, Santa Clara, CA, USA), as described previously [18,24]. IGROV-1 cells were exposed to 1 µM rucaparib, olaparib, niraparib, pamiparib or talazoparib and ES-2 cells to rucaparib, olaparib or niraparib for 1 h before the drug was washed off with PBS and replaced with fresh media prior to cells being harvested immediately or after 1, 24, 48 or 72 h of incubation in a drug-free medium. Cells were permeabilised and PARP activity measured in comparison to the untreated cells, and cells with 1 μM drug were added directly to the reaction mixture. The percentage of PARP activity was calculated relative to untreated cells.