A549 cells (10 cm dishes) were treated with vehicle or SBI-797812 (10 μM) for 4 h as described above. Where indicated, 0.5 mM H2O2 was also added to the cells for 30 min (from 3.5 to 4 h). Cell lysis buffer contained RIPA, NAM (10 mM), trichostatin-A (10 μg ml−1; Sigma-Aldrich), protease inhibitor cocktail (1×, Roche), olaparib (5 μM; Cayman Chemical), ADP-HPD (250 nM; Millipore Sigma) and benzonase nuclease (0.2 U μl−1; Sigma-Aldrich). Where indicated (Fig. 5c ), olaparib and ADP-HPD were excluded during the cell lysis step. Samples were probed by western blotting with the following antibodies: rabbit monoclonal anti-PARP1 (Cell Signaling, cat no. 9532, clone 46D11) 1:1000 dilution, rabbit polyclonal (affinity-purified) anti-PAR (Trevigen 4336-APC-050) 1:1000-dilution, rabbit monoclonal anti-histone H4 (Cell Signaling, cat no. 13919, clone D2X4V) 1:1000-dilution, and rabbit polyclonal (affinity-purified) anti-acetyl-histone H4(Lys16) (Millipore Sigma, cat no. 07-329) 1:5000-dilution. Protein bands were visualized with IRDye-labeled secondary antibodies (LI-COR Biosciences) followed by scanning with the Odyssey Digital Infrared Imaging System (LI-COR Biosciences).
Olaparib
Manufactured by Cayman Chemical
Sourced in United States
Olaparib is a chemical compound used in laboratory research. It functions as a inhibitor of the enzyme poly(ADP-ribose) polymerase (PARP), which is involved in DNA repair. Olaparib is commonly used in studies related to cancer biology and DNA damage response pathways.
Automatically generated - may contain errors
Lab products found in correlation
Azacytidine, by Cayman Chemical (2 mentions)
Methotrexate, by Cayman Chemical (2 mentions)
Cellometer auto 1000, by Revvity (2 mentions)
Rucaparib, by Cayman Chemical (2 mentions)
Benzonase, by Merck Group (2 mentions)
6 thioguanine, by Merck Group (2 mentions)
Hek293t, by Selleck Chemicals (2 mentions)
Meljuso, by Selleck Chemicals (2 mentions)
Vemurafenib s1267, by Selleck Chemicals (2 mentions)
Talazoparib bmn 673, by Selleck Chemicals (2 mentions)
Hek293t, by American Type Culture Collection (2 mentions)
Etoposide, by Cayman Chemical (2 mentions)
Nutlin 3a, by Cayman Chemical (2 mentions)
Pemrametostat, by Selleck Chemicals (2 mentions)
Venetoclax, by Cayman Chemical (2 mentions)
Pinometostat, by Cayman Chemical (2 mentions)
Mivebresib, by Cayman Chemical (2 mentions)
Alvespimycin, by Cayman Chemical (2 mentions)
Glomax explorer multimode microplate reader, by Promega (2 mentions)
Ve 821, by Cayman Chemical (2 mentions)
24 protocols using olaparib
1
Analyzing PARP1 and Histone Acetylation
2
Anticancer Drugs Screening on hESCs
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All 10 anticancer drugs were selected based on their selective function and importance as drugs that are frequently used in the clinic. Ten drug concentrations were tested for each drug. hESC cells were grown on a 96 well plates in a triplicate manner. hESC density was 15,000 cells per well. For each drug, six plates were used to allow six time points per concentration. The drug in each concentration was added on Day ‘0’ and the medium was replaced every 24 h. Cell viability was assessed by a CellTiter‐Glo luminescent cell viability assay according to the manufacturer's instructions (Promega) and cell viability was monitored following 1, 2, 3, 6, 10 and 13 days. Luminescence reads for the target genes were normalized to control conditions, and the replicate experiments were averaged. This comprehensive calibration regime allowed a careful selection of concentrations that produce significant cell death yet allow some cell recovery. All anticancer drugs in this study: Azacytidine, Bleomycin, Vorinostat, Imatinib, Sunitinib, Vemurafenib, Methotrexate, Olaparib, Ibrutinib and Enzalutamide, were purchased from Cayman Chemical (Ann Arbor, Michigan, USA). Preparations of all drugs were done according to vendor protocols.
3
MTT Assay for Cell Viability
4
Olaparib Cytotoxicity on Prostate Cancer Cells
5
Olaparib Dose-Response Assay
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10,000 cells were plated into 60mm plates and dosed with the indicated concentrations of Olaparib (Cayman Chemical Company). Cells were incubated at 37°C and 5% CO2 for 4 days before being trypsinized and counted on a Cellometer Auto 1000 (Nexcelom Bioscience LLC).
6
Investigating Redox Signaling Pathways
7
Quantifying DNA Damage and Micronuclei
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For γH2AX foci and micronuclei analysis, cells were seeded onto glass coverslips and 24 h later pretreated with DMSO or 5 µM olaparib (10621; Cayman Chemical). After 24 h, cells were fixed with 2% (vol/vol) PFA for 15 min, permeabilized with 0.5% Triton X-100 in PBS for 15 min on ice, and blocked with 5% BSA/PBS for 1 h. Coverslips were then incubated with anti-γH2AX antibody (1:1,000) for 1 h at RT. After three washes in PBS, samples were incubated with Alexa Fluor 594 goat anti-mouse IgG (H+L) secondary antibody (A11032; Invitrogen) for 1 h at RT, and slides were mounted with VECTASHIELD mounting medium with DAPI (Vector Laboratories). Cells were imaged using a FluoView 3000 confocal microscope (Olympus) using TruSpectral high-efficiency gallium arsenide phosphide spectral detectors with a 60× plan apochromat oil objective lens with 1.42 NA. Image acquisition was performed with FV-10 ASW3.1 software (Olympus). Quantification of γH2AX foci and micronuclei was performed using ImageJ software.
8
Quantifying Cellular Sensitivity to DNA Damage
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To determine cellular sensitivity to DNA damaging agents, HeLa cells were transfected with the indicated siRNA oligonucleotides. Forty eight hours later, 500 cells were seeded in six-well dishes and treated with the indicated concentrations of Olaparib (Cayman Chemical) or doses of IR. Following 7–10 days in culture, cells were fixed, stained and colonies were counted. Triplicate cultures were scored for each treatment. Shown is the relative survival as compared to the undamaged control and the error bars present the standard error of three independent experiments.
9
THP1 and MEF Cell Stimulation
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THP1 cells were treated with 500 ng/ml Ultrapure LPS from Escherichia coli 0111: B4 (Invivogen, Cat# tlrl-3pelps) or Salmonella minnesota (Invivogen, Cat# tlrl-smlps) in standard RPMI complete media. Similarly, THP1 cells in RMPI complete media were stimulated with 500 ng/ml Pam3CSK4 (Invivogen, Cat# tlrl-pms). Primary MEFs were transfected with 0.5 μg of 5′ppp-dsRNA or 4 μg HT-DNA per well of a 6-well plate using with Lipofectamine 2000 as described earlier117 (link). Dox (Sigma, Cat# D1515) was supplemented in the THP1 and MEF culture media at 0.5–2 μM. CPT (Cayman Cat# 11694) was used at a concentration of 1–5 μM for the indicated time periods. Cells were exposed to 5 Gy IR and harvested 1 h post-IR. THP1 cells were pretreated with 2 μM TBK1 inhibitor MRT67307 (EMD Millipore, Cat# 506306) or dimethyl sulfoxide vehicle control as described earlier118 (link) preceding cGAMP treatment. THP1 cells were stimulated with recombinant human IFN-β (0.5–100 ng/ml) (Peprotech, Cat# 300-02BC) in culture media. Cells were pretreated with 25 μM ATM inhibitor KU-55933 (Sigma Cat# SML1109), 10 μM PARP inhibitor olaparib (Cayman Cat# 10621), or 10 μM PARP1 inhibitor rucaparib (Cayman Cat# 15643) for 2 h. Cells were harvested at the indicated time points for analysis. Cellular NAD+ levels were boosted by preincubating cells for 24 h with 2–4 mM NAM (Sigma Cat# 47865-U).
10
Cell Lysis and Protein Extraction
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For MMS treatment, cells were damaged with 2 mM MMS (Sigma-Aldrich; St. Louis, MO, US) for 1 hr. In case of H2O2, cells were damaged with 2 mM H2O2 (Sigma-Aldrich; St. Louis, MO, US) for 10 min. Cells were lysed as previously described (Fontana et al., 2017 (link)) in the following buffer: 50 mM Tris-HCl pH 8.0, 100 mM NaCl, and 1% Triton X-100. Immediately before lysing the cells, the lysis buffer was supplemented with 5 mM MgCl2, 1 mM DTT, proteases and phosphatases inhibitors (Roche; Basel, Switzerland), 1 μM ADP-HPD (Calbiochem, La Jolla, CA), and 1 μM Olaparib (Cayman Chemical, Ann Arbor, MI). After the cell pellet was resuspended in the supplemented lysis buffer, Benzonase (Sigma-Aldrich; St. Louis, MO, US) was added (Fontana et al., 2017 (link)).
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