Pfm01

T24 and HEK293 cells were grown in DMEM medium, and RT112 cells were grown in RPMI-1640 medium. DR-GFP U2OS cells were provided by Dr. Sovan Sarkar, University of Oxford, and grown in DMEM medium supplemented with 3 μg/mL puromycin. SA-GFP U2OS cells were provided by Dr. Pavel Janscak, University of Zurich, and grown in DMEM supplemented with 3 μg/mL puromycin. Stable HEK293 Flp-In T-REx (p97-E578Q-cSSH) cells for p97 SILAC-MS were used as previously described (Meerang et al., 2011 (link)). RT112 cells with stable shRNA knockdown of MRE11 were kept in RPMI-1640 medium supplemented with G418 (500 μg/mL), and provided by Dr. Juri Na, University of Oxford. Cells were treated as follows: 10 μM BrdU (#B5002; Sigma) and 10 μM Edu (#C1042; Sigma) for 24 hours and in the last 20 minutes of the 24 hours, respectively; 10 μM CB-5083 (#HY-12861; MCE) for 2 hours; 500 μM hydroxyurea (#H8627; Sigma) for 1 hour; MRE11 inhibitors used were 100 μM Mirin (#M9948; Sigma), 100 μM PFM01 (#6222; Tocris), 100 μM PFM39 (#SML1839; Sigma) for 3 hours. Concentrations of PFM01 and PFM39 were chosen according to a previous publication (Shibata et al., 2014 (link)). For clonogenic survival assays, appropriate concentrations of CB-5083 and Mirin were applied for 6 hours and 24 hours, respectively.

mESCs were treated 40–48 h after passage. mESCs were incubated in culture with 250 µM 5-chloro-2′-deoxyuridine (CldU, Sigma, C6891) for 20 min, washed twice with PBS, incubated with 2 mM hydroxyurea (HU, Sigma, H8627) for 3 h, washed twice with PBS, and incubated with 250 µM 5-Iodo-2′-deoxyuridine (IdU, Sigma I7125) for 20 min. Then, 100 µM IAA (Sigma, I5148), 50 µM mirin (Cayman, 13208), 10 µM PFM01 (Tocris, 622210, Bristol, UK), 10 µM DNA2-IN-C5 (Aobious, AOB9082, Gloucester, MA, USA), 2.5 µM CSN5i-3 (Novartis Pharma, Basel, Switzerland), 25 µM compound 33-11 (ChemBridge Corporation, 6655693, San Diego, CA, USA), or 5 µM ML216 (Aobious, AOB1300) were added at the indicated time points. In place of HU treatment, aphidicolin (Cayman Chemical, 14007) was used at 15 µM. Labeled mESCs were treated with TrypLE and resuspended in PBS at 2 × 10⁵ cells/mL. DNA fiber spreading and immunostaining were performed as previously described [137 (link)]. Primary antibodies used were rat anti-BrdU (CldU) (Abcam, Waltham, USA) and mouse anti-BrdU (IdU) (Becton Dickinson, Franklin Lakes, NJ, USA). Secondary antibodies used were Alexa Fluor anti-rat 568 and Alexa Fluor anti-mouse 488. Antibody information is presented in Supplementary Table S1.

DNA2 inhibitor (NIH, Developmental Therapeutics Program, Cat# NSC-105808) was dissolved in dimethylsulfoxid (DMSO) at 10 mM and used at a final concentration of 4 µM. Mirin, an MRE11 inhibitor (2-amino-5-[(4-hydroxyphenyl) methylene]-4(5H)-thiazolone, Santa Cruz Biotechnology) was dissolved in DMSO at 100 mM and was used at 75 µM final concentration. PFM01, a specific inhibitor of MRE11 endonuclease function ((5Z)-5-[(4-Hydroxyphenyl) methylene]-3-(2-methylpropyl)-2-thioxo-4-thiazolidinone, Tocris) was dissolved in DMSO at a 50 mM concentration and was used at a final concentration of 10 μM. In the experiments presented here these two MRE11 inhibitors were used in combination. NU7441 (8-(4-Dibenzothienyl)-2-(4-morpholinyl)-4H-1-benzopyran-4-one, Selleckchem), a specific DNA-PKcs inhibitor was dissolved in DMSO at a final concentration of 10 mM and was used at a working concentration of 10 µM. Unless indicated otherwise, all inhibitors were added to the cell cultures 1 h before irradiation and were maintained until collecting cells for analysis.