A4487

Hydroxyurea (3 mM for 2 h for QIBC and immunoblotting, 0.5 mM, 1 mM, or 2 mM for 24 h for clonogenic assays, H8627; Sigma-Aldrich), UCN-01 (300 nM for 1 h, U6508; Sigma-Aldrich), APH (50 μg/ml for 2 h, A4487; Sigma-Aldrich), thymidine (2.2 mM for 12 or 17 h, T1895; Sigma-Aldrich), nocodazole (100 ng/ml for 12 h, M1404; Sigma-Aldrich), PD0332991 (5 μM for 12 h, PZ0383; Sigma-Aldrich), TAK-931 (300 nM for 12 h, HY-10088; Biotech), bleomycin (25 μg/μl for 3 h on and 3 h off, S1214; Stratech), CPT (500 nM for 6 h, C9911; Sigma-Aldrich), Z-VAD-FMK (50 μM, ab120487; Abcam). Protein expression was induced in with 0.5–1 μg/ml tetracycline for 24 h (T7660; Sigma-Aldrich).

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)).