Sml0364

Custom anti-RNF4 antibody was generated as previously described (79 (link)). Commercial antibodies used in this study included: SUMO2/3 (ab3742, Abcam), p53 (2524, Cell Signaling Technology [CST]), cleaved caspase-3 (9661, CST), tubulin (T8238, Sigma-Aldrich), KAP1–p-S824 (A300-767A, Bethyl), CHK1–p-S345 (2341, CST), CHK1 (sc-8408, Santa Cruz Biotechnology), γ-H2AX (05-636, Millipore), γ-H2AX–Alexa Fluor 488 (20304S, CST), histone H3 (ab1791, Abcam), TOP2a (sc-365916, Santa Cruz Biotechnology), TOP2b (MAB6348, Novus), PARP1 (9542, CST), RNF10 (16936-1-AP, Proteintech). Antibody specific for mouse POLD4 was provided by Marietta Lee (New York Medical College, Valhalla, New York, USA). The following chemicals were used: olaparib (S1060, Selleckchem), hydroxyurea (H8627, Sigma-Aldrich), aphidicolin (A4487, Sigma-Aldrich), cisplatin (S1166, Selleckchem), MMS (129925, Sigma-Aldrich), gemcitabine (G6423, Sigma-Aldrich), P22077 (662142, Sigma-Aldrich), P5091 (2277, Biovision), 2-D08 (SML1052, Sigma-Aldrich), ML-792 (407886, MedKoo), RI-1 (SML1294, Sigma-Aldrich), and B02 (SML0364, Sigma-Aldrich). For plasmids, human RAD51 cDNA was inserted into pRK5-FLAG, and human RNF4 cDNA was inserted into pMX-PIE-IRES-EGFP. Amino acid substitutions for RNF4-CS and RNF4-ΔSIM were performed as previously described (78 (link), 80 (link)).

For different inhibitor assays, the cells were pre-treated with mirin (Sigma, M9948-5MG; 25 µM), B02 (Sigma, SML0364; 10 µM) or RI-1 (Sigma, 553514-10MG-M; 1 µM) for 16 h. For the cell-cycle arrest experiment, the cells were pre-treated with thymidine (Sigma, T9250-1G, 2 mM) for 18 h, the thymidine was removed, the cells were cultured in normal DMEM media with 10% FBS without thymidine for 9 h and thymidine was added to the cells (final concentration of 2 mM) for a second round of 18 h. For the DTB–mirin/RI-1/B02 groups, mirin (25 µM), B02 (10 µM) or RI-1 (1 µM) were added to the cells with the second treatment round with thymidine (2 mM). After the inhibitor and thymidine treatment, the cells were transfected using Lipofectamine 3000 following the manufacturer’s instructions. The cells were analysed on a CytoFLEX flow cytometer 3 d later.