Ku55933

T-DXd was provided by Daiichi Sankyo Co., Ltd. (Tokyo, Japan). Other reagents used in this study were acquired from the indicated suppliers: trastuzumab (Chugai Pharmaceutical Corporation, Tokyo, Japan); KU-55933, VE-821, and UCN-01 (Merck Sigma-Aldrich, St. Louis, MO, USA); IFN-γ (R&D systems, Minneapolis, MN, USA); irinotecan (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan); ultra-LEAF purified human IgG1 isotype control recombinant antibody (BioLegend, San Diego, CA, USA); FITC mouse anti-human HER-2/neu (340553), FITC mouse IgG1 κ isotype control (555909), and 7-AAD (559925) (BD Biosciences, San Jose, CA, USA); PE anti-human HLA-ABC monoclonal antibody (W6/32) (12-9983-42), Alexa Fluor 488 anti-human CD326 (EpCAM) (53-8326-41), and PE mouse IgG2a κ isotype control (eBM2a) (12-4724-81) (Thermo Fisher Scientific, Waltham, MA, USA); HER2/ErbB2 rabbit mAb (#4290), Phospho-Akt rabbit mAb(#4060), Akt (pan) rabbit mAb (#4691), Phospho-ERK1/2 rabbit mAb (#4370), and ERK1/2 rabbit mAb (#4695) (Cell Signaling Technology, Danvers, MA, USA); anti-human HLA class I (HLA-ABC) mAb (D367-3) (Medical & Biological Laboratory, Aichi, Japan); Anti-β-actin antibody (sc-69879) (Santa Cruz Biotechnology, Dallas, TX, USA).

ATM inhibitor KU55933 (Sigma-Aldrich, Misuri, USA), ATR inhibitor VE-821 (ApexBio, Houston, USA), and DNA-PK inhibitor NU7441 (ApexBio) were dissolved in DMSO to obtain concentrated stocks. DDR inhibition was carried out by pretreating cells with the indicated final concentration of each inhibitor for 2 h and keeping the inhibitor present during the entire infection assay. To induce DNA damage, cells were treated with neocarzinostatin (NCS; 0.5 mg/mL, Sigma-Aldrich) for 30 min.

Generally, 2 × 10⁵ cells were seeded per well of a 12-well plate and infected the next day at the indicated multiplicity of infection (MOI). Cells from separate wells were trypsinized and counted to determine cell number. Virus inoculum was prepared in PBS supplemented with 0.3% BSA, 1 mM Ca²⁺/Mg²⁺, 100 units/mL penicillin, and 100 μg/mL streptomycin. Cells were incubated with the inoculum for 1 h, washed 3 times with PBS, and then overlaid with DMEM supplemented with 0.1% FBS, 0.3% BSA, 20 mM Hepes, 100 units/mL penicillin, and 100 μg/mL streptomycin. Where indicated, IFNα2a (Roferon-A; Roche), etoposide (Sigma-Aldrich; E1383), KU55933 (Sigma-Aldrich; SML1109), MG132 (Sigma-Aldrich; M7449), lactacystin (Enzo Life Sciences; BML-PI104-0200), ruxolitinib (Santa Cruz; sc-364729), or BX-795 (Sigma-Aldrich; SML0694) was added to the medium at the indicated concentration. Virus titers in the supernatants at the indicated time points were determined by standard plaque assay on MDCK cells. All infections with IBV were performed at 33 °C, while 37 °C was used for all other conditions.

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).