Mirin

Human colon carcinoma BE cells (diploid cells that contain an oncogenic Kras-G13D mutation as well as the BRAF-G463V oncogenic mutation; a gift from Simon Wilkinson and Christopher Marshall, Institute of Cancer Research, London, UK; Petsalaki et al., 2018b (link)), cervical carcinoma HeLa cells stably expressing LAP2b fused to RFP (a gift from Daniel Gerlich, Institute of Molecular Biotechnology, Vienna, Austria; Steigemann et al., 2009 (link)), or HeLa cells stably expressing α-tubulin fused to GFP (a gift from Jan-Michael Peters, Institute of Molecular Pathology, Vienna, Austria) were grown in DMEM (Gibco) containing 10% FBS at 37°C in 5% CO₂. Cells were treated with 10 µM Chk2 inhibitor II (C3742; Millipore Sigma), 10 µM KU-55933 (sc-202963; Santa Cruz Biotechnology), 1 mM hydroxyurea (Sigma), 10 µM etoposide (Sigma), or 25 µM mirin (ab141182; Abcam) as appropriate. siRNA duplexes were transfected into BE cells 24 h before analysis using Lipofectamine 2000 (Invitrogen). For expression of GFP proteins, plasmids were transfected into cells in the absence or presence of appropriate siRNA duplexes 24 h before analysis or further drug treatment using Turbofect (Thermo Fisher Scientific). All cell lines used exhibited consistent morphology and growth properties and were negative for mycoplasma contamination.

Antibodies against IKKβ, catalase, SOD1, p21, p27, p-Chk1(S345), Chk1, p-Chk2(T68), Chk2, p-H2AX(S139), H2AX, p-ATM(S1981), ATM, MGMT, PARP, Caspase-3, p-p53(S15), p53, survivin, XIAP, cIAP-1, cIAP-2, Mre11, Rad50, p95/NBS1, were purchased from Cell Signaling (Beverly, MA). Antibodies against Bak, Bcl-2, Bcl-xL, GAPDH, and actinin were purchased from Santa Cruz Biotechnology, Inc. Fotemustine and antibody against Flag (M2) was purchased from Sigma. 8-OHdG ELISA kit, mirin and SC-514 for in vivo experiment were purchased from Abcam. SC-514 for in vitro experiment, carmustine and lomustine were purchased from Enzo Life Sciences. PLX4032, BMS-345541, TPCA-1, LY-2409881, AZD3264, IKK-16 and PS-1145 were purchased form Selleckchem ML 120B dihydrochloride and PF184 were purchased from Tocris Bioscience. Crystal violet, propidium iodide (PI), N-acetyl-L-cysteine (NAC), temozolomide, 3-Amino-1,2,4-triazole (ATZ), hydrogen peroxide solution and 3-(4,5-Dimethyl-2-thiazolyl)−2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma. 6-Carboxy-2′,7′-dichlorofluorescein diacetate (DCF) was purchased from Thermo Scientific.

Doxycycline (Dox, Sigma-Aldrich) was added to cell culture at 1 μg/ml 24h before irradiation. Mirin (MRE11 exonuclease inhibitor, Abcam) was added to cell culture at 100 μM 10 min before irradiation. KU55933 (ATM inhibitor, Abcam) was added to cell culture at 10 μM 10 min before the addition of Neocarzinostatin. Neocarzinostatin (Sigma-Aldrich) was added to cell culture at 150 ng/ml.