Olaparib

For oxidative stress induction, HeLa cells were treated with 300 μM or 1 mM hydrogen peroxide (H₂O₂) or 300 μM sodium arsenite for the indicated period of time.
For induction of SGs in nucleus, cells were treated with 4 μM actinomycin D (ActD) (Thermo Fisher Scientific, #11805017) for 1 h before treatment with 300 µM H₂O₂.
For induction of SGs in cytoplasm, cells were treated for 60 min with 300 µM sodium arsenite or 200 µM puromycin (Sigma, St. Louis, MO, USA, #P9620) for 1 h before treatment with 300 µM H₂O₂.
For inhibition of PARP activity, cells were pre-treated with 10 μM olaparib (Apexbio Technology, Houston, TX, USA, #A4154) or 1 μM talazoparib (MedChemexpress, #HY-108413) and diluted in DMSO for 1 h before and during indicated treatments.
To study the disassembly of SGs formed by treatment with sodium arsenite, cells were treated with 300 µM sodium arsenite for 60 min, then the culture medium was replaced with fresh medium containing DMSO (control), 300 µM H₂O₂ or 3 µM olaparib or 300 µM H₂O₂, as indicated, and then the cells were left alone for one hour for further fixation and analysis.
After incubation with the drugs at indicated time points, the cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) in PBS for 45 min at 37 °C, if another cell fixation method is not indicated.

Selection of the gene-edited population was done either 24 h post electroporation by puromycin (for 72 h; 0.4, 0.7 and 1.1 µM; EMD Millipore, Billerica, MA, USA) or 72 h post electroporation by PARPi (for 7 days; olaparib (0.1, 1 µM; ApexBio, Houston, TX, USA), KU0058948 (0.1, 1 µM; Axon Medchem, Reston, VA, USA)). Monoclonal populations were prepared by plating the gene-edited cells at low density (300 cells per 10-cm dish). Forty-eight hours later, glass-cloning cylinders were placed around single-cell colonies using sterile silicon grease. Clonal populations were progressively transferred into larger wells/flasks. Selected cells were analyzed by PCR. For HDR donor specific detection: F: 5′-TGAAGAAGCTTCCGAAACCG-3′ and R: 5′-GCCACACAGTGCACCATAGA-3′. For locus amplification and subsequent donor detection by HindIII digest the following primers were used: PCR F: 5′-CACCAAGCCATATCTTACCACC-3′, PCR R: 5′-ACAGCAGAGTTTCACAGGAAGT-3′. PCR was performed at: 95 °C × 2 min, 40 cycles of 95 °C × 45 s, 57 °C × 45 s, and 68 °C × 1 min.

Inhibitors (PARP inhibitor (Pi) 100 μM NU1025 (Sigma) or 20 μM olaparib (Apexbio Technology), 10 μM DNA–PKcs inhibitor (Di) NU7026 (Sigma), 10 μM ATM inhibitor (Ai) KU55933 (Calbiochem), 1 μM PARG inhibitor (PARGi) DEA ((6,9-diamino-2-ethoxyacridine lactate monohydrate) (Trevigen)) were added to the cell culture one hour prior to damage induction. DMSO only was added to control cells.

Cell viability was determined by counting the cells stained with the Hoechst 33342 nucleic acid stain solution at 24 h increments over a 96 h period. Briefly, glioblastoma cell lines U87MG, U251MG, and U373MG transfected with shRNAs to RAD51 or Scm control, were seeded at a density of 1000 cells/well in a 96-well plate. Wells were treated with either DMSO (control) or the PARPi olaparib (AZD2281, Ku-0059436, Apexbio, cat# A4154) at a concentration of 1 μM and the cells were incubated at 37 °C (with 5% CO₂) for 96 h. At each 24 h interval, Hoechst 33342 (Life Technologies, cat# H1399) at a concentration of 0.2 μg/mL was applied to a subset of wells and the subsequently-labeled cell nuclei were counted and analyzed via Cytation 5 and Gen5. Experimental assays were repeated in triplicate with 3 wells per cell treatment/time-point per experiment (n = 3, N = 9). Cell growth rate was determined relative to the baseline cell count performed upon cell seeding (Day 1). Statistical analysis was performed using the 2-way ANOVA using the Tukey’s post hoc test with no correction.