Ku55933

HeLa cells were transfected with scrambled siRNA control oligos or siRNA oligos specifically against TRAF6 (Bioland) using Lipofectamine 2000 transfection reagent (Thermo Fisher) or transfected with scrambled control shRNA or shRNA pLV-EGFP:T2A:Puro-U6-hTRAF6 from VectorBuilder (https://en.vectorbuilder.com/). After 48–72 h, the cells were harvested and lysed. Knockdown efficiency was confirmed using quantitative RT-PCR. hTRAF6 siRNA targeting sequences: GCAGUGCAAUGGAAUUUAUTT, CCCAGUCACACAUGAGAAUTT and GCAAAUGUCAUCUGUGAAUTT. TRAF6 shRNA target sequence: AGCGCTGTGCAAACTATATAT.
Cycloheximide (CHX, 50 μg/ml, purity: 95%) or MG132 (1 μM, purity: >90%) was added to the cell media for 0−24 h to inhibit protein synthesis or degradation, respectively. CPT (1 μM; purity: 99%; Sigma) or HU (1 mM; purity: 98%; Sigma) was added to the cell media for 4 or 16 h, respectively, before cell collection. The TRAF6 E3 ligase inhibitor C25–140 (ChemDiv, Catalog #G827–0140) was added for 48 h at a final concentration of 30 μM. The TRAF6 inhibitory peptide T6PD (Novus Biologicals, Catalog #NBP2–26506) was added for 24 h at a final concentration of 100 μM. The ataxia-telangiectasia mutated (ATM) inhibitor KU-55933 (Abcam, Catalog #ab120637) was added for 12 h at a final concentration of 10 μM.

HEK293T (CRL-3216), HCT116 (CCL-247) and U2OS (HTB-96) cell lines were purchased from ATCC and cultured in Dulbecco’s Modified Eagle’s Medium or McCoy’s 5A supplemented with 10% fetal bovine serum (FBS) at 37 °C in 5% (v/v) CO₂.The following inhibitors were used: VE-822 (Selleckchem: s7102), NU7441 (selleckchem:S2638), and KU55933 (abcam: ab120637).

Indicated cells were cultured on coverslips and treated with 50 µM cisplatin (Sigma, P4394), 100 µM olaparib (LC Laboratories, O-9201), 10 µM DNA-PK inhibitor AZD7648 (ChemScene, CS-0091859), 10 µM ATM inhibitor KU55933 (Abcam, ab120637) or 10 µM ATR inhibitor VX970 (Selleckchem, S7102) for 6 h. After washing with PBS, cells were fixed in 3% paraformaldehyde for 15 min and permeabilized in 0.5% triton X-100 solution for 5 min at room temperature. Cells were then blocked with 5% goat serum and incubated with indicated primary NPM1 (Invitrogen, 32-5200) or γH2AX (CST, 9718 S) antibody overnight in 4 °C. Subsequently, samples were washed and incubated with Alexa Flour labeled secondary antibody for 60 min. DAPI staining was performed to visualize nuclear DNA. The coverslips were mounted onto glass slides with anti-fade solution and visualized using a Nikon ECLIPSE E800 fluorescence microscope. For OVCAR-1 CP cells and PEO-1R cells, they were pretreated with lovastain (5 μM), farlutin (5 µM) or slinitasertib (5 μM) for 36 h, followed by 50 µM cisplatin (Sigma, P4394) and 100 µM olaparib (LC Laboratories, O-9201) for 6 h, respectively. Then they were analyzed by immunofluorescence staining and confocal imaging as mentioned above.