Olaparib

This study used two human osteosarcoma cell lines, U2OS (Korean Cell Line Bank, Seoul, Korea) and KHOS/NP. KHOS/NP cells were kindly provided by Chang-Bae Kong (Department of Orthopedic Surgery, Korea Institute of Radiological and Medical Science). The cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco BRL, Gaithersburg, MD) containing 10% FBS (Gibco BRL) and 1% penicillin/streptomycin (100 U/mL) at 37 °C in a humidified incubator under 5% CO₂. This study used doxorubicin (D1515, Sigma, St. Louis, MO), KU-55,933, an ATM inhibitor, (SML1109, Sigma), and olaparib, a PARP inhibitor (SC-302,017, Santa Cruz Biotechnology, Santa Cruz, CA). The vector for SIRT6-specific shRNA was purchased from GenePharma Co. (GenePharma, Shanghai, China). The SIRT6 duplex had the sense and antisense sequences 5′-CACCGCTACGTTGACGAGGTCATGATTCAAGAGATCATGACCTCGTCAACGTAGCTTTTTTG-3′ and 5′-GATCCAAAAAAGCTACGTTGACGAGGTCATGATCTCTTGAATCATGACCTCGTCAACGTAGC-3′, respectively [16 (link)]. A pFLAG-CMV-2 plasmid vector was used as a control vector. The vector overexpressing wild-type SIRT6 (pFLAG2_SIRT6) was synthesized by Cosmogenetech Co. Ltd. (Cosmogenetech Co. Ltd, Seoul, Korea) [16 (link)]. Transfection was performed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA).

Monoclonal anti-MGMT antibodies were obtained from LTK BioLaboratories (Taoyuan, Taiwan). Chemical agents including O6-benzylguanine (O6BG) and olaparib and antibodies targeting BRCA1, pBRCA1 (Ser988), BRCA2, RAD51, β-actin, and lamin A/C were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Monoclonal anti-γ-H2AX antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Other experimental reagents used are listed in a previous report [18 (link)].

AQ4N (Sigma) and KP167 were reconstituted in distilled water to a stock concentration of 10 mM. Olaparib (Santa Cruz Biotechnology) and KU‐55933 (Tocris Biosciences) were reconstituted in DMSO to a stock concentration of 10 mM. All stock solutions were aliquoted and stored at −20°C for a maximum of 3 months and repeat freeze thaw cycles avoided. A new batch of drugs was always tested for similar response between new and old to ensure consistency and to minimize batch–batch variability.