Neocarzinostatin ncs

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.

The HCT116-TP53^−/− [30 ] and RKO-TP53^−/− cell lines [34 (link)] were generated in the lab of Bert Vogelstein (Johns Hopkins School of Medicine, Baltimore, Maryland, USA). HCT116 and RKO cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) containing 10% foetal bovine serum (Sigma-Aldrich), 2 mM L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin (all PAN Biotech) at 37°C in 5% CO₂. Cells were seeded 1 or 2 days before the experiment in 10-cm dishes and grown to 60–80% confluency. Cells were then treated with neocarzinostatin (NCS, Sigma-Aldrich) at a concentration of 0.2 µg/ml for 4 h (HCT116 cells) or 0.4 µg/ml for 5.5 h (RKO cells). For control, cells were treated with an equal volume of phosphate buffered saline (PBS). During puromycin incorporation assays, cells were treated with puromycin (Gibco) at a concentration of 1 µg/ml, or H₂O alone as control, for 5 min prior to cell lysis.

DharmaFECT1 transfection reagent was obtained from Dharmacon (GE Dharmacon, Lafayette, CO, USA). Neocarzinostatin (NCS) was obtained from Sigma–Aldrich (St. Louis, MO, USA). The ATM inhibitor, KU55933 (46 (link)), the DNA-PK inhibitor, NU7441 (47 (link)) and the ATR inhibitor, AZ20 (48 (link)) were obtained from Tcris Bioscience (Bristol, UK). Anti-53BP1 mouse monoclonal antibody was a generous gift from T. Halazonetis, anti-53BP1 polyclonal antibody was obtained from Novus Biologicals, LLC (Littleton CO, USA), anti-pS139-H2AX monoclonal antibody was obtained from Merck Millipore (Darmstadt, Germany), anti-PABPN1 and anti-BRCA1 antibodies were obtained from Abcam (Cambridge, MA, USA), phospho-antibodies against phosphorylated Ser 95 of PABPN1 and phosphorylated KAP-1 were obtained from Bethyl Laboratories, Inc. (Montgomery, TX, USA), anti-ATM obtained from Sigma–Aldrich (St. Louis, MO, USA), anti-HSC70 monoclonal antibody was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), anti-RAD51 was obtained from Chalbiochem, Inc. (San Diego, CA, USA), antimouse and anti-rabbit IgG, Alexa 488/568/633 were purchased from Molecular Probes (Leiden, Netherlands), and HRP-conjugated anti-rabbit IgG and anti-mouse IgG were obtained from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA, USA). Immunoblotting was carried out as previously described (49 (link)).

The following antibodies and reagents were used: anti–phospho-Ser1981-ATM, anti-phospho-Chk2(T68), anti-Chk2, anti-phopsho-S15-p53, anti-PARP1 (all from Cell Signaling, Beverly, MA, USA); anti-ATM (2C1), anti-p53 (DO-1), anti-HSP90 (F8), anti-actin B, anti-vinculin, anti-GAPDH (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-PAR (Ab-1; Sigma Aldrich, St. Louis, MO, USA, cat no AM80); anti-phospho-S824 Kap1 (Abcam, Cambridge, UK, ab70369); anti-SAM68 (purified); neocarzinostatin (NCS) (Sigma-Aldrich, St. Louis, MO, USA, cat no N9162), a radiomimetic chemotherapy drug, was used at 500 ng/mL concentration, and KU55933 (Sigma Aldrich, St. Louis, MO, USA, cat no SML11109), a specific inhibitor of ATM kinase, was used at 10 μM concentration.

HeLaS3 cells grown on 13 mm–diameter round cover glasses (Matsunami) were arrested at M phase by treatment with 0.1 µg/ml nocodazole (Wako) for 3 h. Etoposide (Sigma-Aldrich) or neocarzinostatin (NCS, Sigma-Aldrich) or ICRF-159 (Sigma-Aldrich) was added to the culture medium to a final concentration of 10 µM, 1 ng/ml, 10 µM, respectively. Cells were incubated for 15 min in medium containing the drug and then washed twice with PBS. For detection of anaphase bridges, Etoposide-treated cells were incubated in fresh culture medium for 1 h, fixed with 4% (w/v) paraformaldehyde (Sigma-Aldrich), and stained with 4′,6-Diamidino-2-Phenylindole (DAPI). The frequency of cells with at least one bridge was calculated by dividing the number of cells containing bridges by the number of total anaphase cells.