Ag14361

The PARP inhibitors used in this study were purchased from Selleckchem (Houston, TX, USA) with the following names and catalog numbers: A-966492 (catalog number S2197), AG14361 (catalog number S2178), AZD2461 (catalog number S7029), E7449 (catalog number S8419), G007-LK (catalog number S7239), Niraparib (MK-4827; catalog number S2741), NMS-P118 (catalog number S8363), NU1025 (catalog number S7730), Olaparib (AZD2281, Ku-0059436; catalog number S1060), PJ34-HCl (catalog number S7300), Rucaparib (AG-014699, PF-01367338; catalog number S1098), Talazoparib (BMN673; catalog number S7048), and UPF1069 (catalog number S8038). Stock solutions were dissolved in dimethyl sulfoxide (DMSO) according to the manufacturer’s protocol to a concentration of 10 mM and stored at −20 °C. Table 1 lists the PARP inhibitors used in this study and their proposed targets.

The medium was removed and replaced with fresh medium containing or not, either 1-methyl-4-phenyl-pyrididium (MPP⁺, at 2, 4, 8 and 16 μmol/L), a metabolite of MPTP, 6OHDA (at 5, 10, 20 and 30 μmol/L) or rotenone (0.1, 1, 10 and 20 nmol/L) (all from Sigma Aldrich) at different concentrations, for different times of incubation (6 wells per condition). Here, these three toxins were used to mimic in cell cultures in vitro cytopathic effects observed in the brains of PD-suffering patients. They were dissolved in the defined culture media mentioned above. Involvement of poly-ADP-ribose polymerase (PARP-1), a partner involved in necroptosis, was assessed using AG14361 (Selleckchem, Houston, Texas, USA), a specific inhibitor of PARP-1 (1 μmol/L, added to the cultures 1 h before DA-toxins).

The human ovarian serous cystadenocarcinoma cell lines UWB1.289, SNU-251, A2780 and SKOV3 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a humidified incubator with 5% CO₂ at 37° C. Oxamate (a specific LDH-A inhibitor), AG14361 (a specific PARP1 inhibitor) and olaparib (a PARP1/2 inhibitor) were obtained from Selleck Chemicals (Houston, TX, USA). The study was reviewed and approved by the Institutional Review Board and the Research Ethics Committee of Shanghai General Hospital.

Ovsaho cells were obtained from JCRB (Japanese Collection of Research Bioresources Cell Bank) cell bank (NIBIOHN, Cell No. JCRB 1046) and grown in MCDB105/199 medium with 10% FBS (Fisher, #10438026) and substituted with 1% Penicillin-Streptomycin. All cells were free of Mycoplasma and their identity was verified by whole exome sequencing at the Broad Institute. For dose-response curves, cells were treated for 72 h with DMSO or the indicated doses of the PARP1 inhibitor AG-14361 (Selleckchem, #S2178), FAS/FASN inhibitor TVB-2640 (Selleckchem, #S9714), Bortezomib (Selleckchem, #S1013), proTAME (Selleckchem, #S9605), GC7 (Sigma Aldrich #259545). In combination assays, inhibitor concentrations were combined in a 1:1 ratio for indicated total concentration. Cell viability was measured using the software in a live-imaging IncuCyte station by counting live cells. Live cells were labeled by adding the Incucyte NucLight Rapid Red (NRR) Reagent (Essen Bioscience, #4717, 1:4000) to the medium. Datapoints of three biological experiments and three technical replicates (total n = 9) were merged and fitted using a non-linear log(inhibitor) versus normalized response with variable slope for dose-response curves using Prism (GraphPad). Combination Index (CI) was calculated based on Chou-Talaly method [53 (link)].