Nu7441

We obtained the human neuroblastoma cell lines SKN-BE, NBLS, IMR32, SMS-SAN, and CHP134 from the American Type Culture Collection (Manassas, VA, USA) and the RIKEN Bioresource Cell Bank, Tohoku University Cell Resource Center (Miyagi, Japan) [19 (link),32 (link),33 (link)]. All cell lines were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 50 µg/mL penicillin, and 50 µg/mL streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). We purchased NU7441, a selective inhibitor of DNA-PKcs, from Sigma Aldrich (St. Louis, MO, USA).

AsiSI-MEFs were described before^{28 (link)}. WT MEFs were obtained from R. Baer (Columbia University). MEFs (WT MEFs and AsiSI-MEFs) were cultured in high-glucose Dulbecco’s modified Eagle’s medium supplemented with l-glutamine, 10% fetal bovine serum and 1% penicillin-streptomycin. ER-AsiSI-MEF cell lines were developed as previously described^{28 (link)}. Cells were treated with doxycycline (Sigma-Aldrich D3072, 3 μg ml⁻¹) for 24 h to induce AsiSI expression. 4OHT (Sigma-Aldrich H7904, 1 μg ml⁻¹) was added for the last 6 h of doxycycline treatment to induce AsiSI translocation. Cells were cotreated with DMSO, CK-666 (Sigma-Aldrich SML-006, 100 μM), NU7441 (Selleckchem S2638, 10 μM), or wiskostatin (Sigma-Aldrich W2270, 3 μM) and incubated at 37 °C for 6 h. For the DNA-PKc inhibitor experiments, cells were pretreated with 10 μM NU7441 for 1 h before induction of damage with 4OHT. MEF cells were transfected with plasmids using Neon Transfection System (1,350 V, 30 ms, one pulse). For flag-actin experiments, AsiSI-MEF cells were transfected with Flag-actin^R62D–NLS 24 h before induction of AsiSI expression. For cas9 experiments, WT MEF cells were transfected with single-guide RNA (5′-CCCTGTCCCAGCGATCGCGC-3′) targeting Chr. 2 48 h before cell lysis.

Cell lines were purchased from the Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan) and maintained in cell culture media (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Gibco). HEK293T human embryonic kidney cells (BCRC 60019) and MCF-7 human breast cancer cells (BCRC 60436) were maintained in DMEM. Chinese hamster ovary (CHO) cells AA8 (BCRC 60126), EM9 (BCRC 60500), and UV24 (BCRC 60175) were maintained in MEM. BO-1055 was synthesized as previously described [19 (link)]. Alkylating agents, including methyl methanesulfonate (MMS), MMC, BCNU and melphalan, inhibitors O⁶-benzylguanine (O⁶-BG), NU6027 and NU7441, as well as DNA strand breaks agent doxorubicin, were purchased from Sigma-Aldrich. KU55933 was purchased from Tocris Bioscience. For DDR induction, BO-1055 or MMC was added to the culture medium for the indicated time period before cells were harvested. Cells irradiated with UV damage (CL-1000; UVP) at 10 J/m² were served as DDR positive controls.

The ATRi VE822 and the EZH2 inhibitor GSK-126 were purchased from Selleck, and the ATM inhibitor KU55933 and the DNA-PK inhibitor NU7441 from Merck Millipore. The Mre11 inhibitor Mirin, 5-bromo-2′-deoxyuridine (BrdU), 5-chloro-2′-deoxyuridine (CldU), 5-iodo-2′-deoxyuridine (IdU) and chloroquine (CQ) were purchased from Sigma-Aldrich. Cisplatin was purchased from Wako, and camptothecin (CPT) was purchased from Abcam.