Dna pk inhibitor nu7441

Human cell lines were all obtained from ATCC more than two years ago and were therefore authenticated using 8-locus STR profiling (LGC standards). Human U2OS osteosarcoma cells were last authenticated in April 2013. H1299 lung carcinoma cells were last authenticated in March 2011 and have not been cultured since. BxPC3 pancreatic adenocarcinoma cells, MCF7 breast cancer cells and OVCAR3 human ovarian cancer cells were last authenticated in April 2014.
VC8 and VC8-B2 cells were obtained from Malgorzata Z. Zdzienicka (16 (link), authentication not available). Cells were confirmed Mycoplasma-free and grown in DMEM with 10% FCS in a humidified atmosphere containing 5% CO₂. OVCAR3 cells were grown in DMEM with 10% foetal bovine serum, 0.01 mg/ml insulin and 1% non-essential amino acids (Sigma). Gemcitabine (Tocris Bioscience) was used at 2 or 5 μM for 2 h. DNA-PK inhibitor NU7441 (Tocris Bioscience) was used at 1 μM. BLM inhibitor ML216 (Sigma-Aldrich) was used at 1.8 μM as previously described (17 (link)).

MRC5-SV40 and HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 μg/mL penicillin, 100 μg/mL and streptomycin. Transient transfections into human cells were performed using GenJet (SignaGen Laboratories) for MRC5-SV40 and PEI (polyethylenimine, Sigma) for HEK293T. Transfections for siRNA were carried out with lipofectamine RNAiMax (Invitrogen) following the manufacturer’s instructions. The siRNAs were siNT: non-targeting pool (Dharmacon) and siRAD18: ACUCAGUGUCCAACUUGCU (Sigma). DNA-PK inhibitor (NU7441, Tocris Bioscience) was used at 2.5 μM 6 h prior to harvesting cells for flow cytometry. NAC (N-acetyl-cysteine, Sigma) treatment was performed twice, with a final concentration of 5 mM, post-24hr and −48hr transfection. To create inducible stable clones to verify DNMT1 and PCNA interaction, GFP-tubulin, GFP-DNMT1 and GFP-DNMT1-ΔPBD cDNAs were cloned into pcDNA5/FRT/TO/Intron vector (Invitrogen, CA). Inducible HEK293T FlpIn Trex GFP-tubulin, GFP-DNMT1 and GFP-DNMT1-ΔBD cells were generated followed by manufacturer’s protocol and were cultured in the same normal medium with 15μg/ml Blasticidin and 80μg/ml hygromycin. Doxycycline (Sigma) was added to medium to trigger the production of GFP fusions.