Nu7441

Lung fibroblasts were cultured in complete medium at 37 °C and 10% CO₂. Medium was changed twice a week and cells were passaged when 70–90% confluency was reached. For transcriptomic experiments, 2.5 × 10⁵ lung fibroblasts per well were plated in complete medium into a 6-well plate overnight at 37 °C and 10% CO₂. Cells were then stimulated with 10 μM CpG (Hycult Biotech) and/or treated with 500 nM Nu7441 (Selleckchem.com) for 24 h (acute) or once every 3 days for a total of 25 days. After treatment, images of the cultured cells were acquired using an EVOS FL inverted microscope at 20x magnification (Thermo Fisher Scientific). Cells were then trypsinized for Flow Cytometry or lysed by Trizol™ (Thermo-Fisher Scientific) for qPCR analysis as described. For studies assessing Collagen 1, IL-8, IL-1ß and αSMA protein expression, cells were then stimulated with 10 μM CpG (Hycult Biotech), 20 ng/ml recombinant TGFß1 (R&D systems) and/or treated with 500 nM of Nu7441 (Selleckchem.com) for 24 or 72 h.

HeLa cells were transduced with five different lentiviral vectors, scrambled shRNA, 53BP1 shRNA (TRCN0000018866, Sigma), Cas9-gRNA, Cas9-DN1S-gRNA, dCas9-DN1S-gRNA and DN1S, or treated with 10 µM NU7441 (Selleckchem) for 20 h. Cells transduced with empty lentiviral vectors were used as controls. Cells transduced with 53BP1 shRNA were selected in puromycin (final concentration: 5 µg/ml). Cells transduced with Cas9-gRNA, Cas9-DN1S-gRNA, dCas9-DN1S-gRNA and DN1S lentivirus vectors, all encoding the mCherry fluorochrome, were sorted for mCherry positive cells. NU7441 treated cells, puromycin selected cells, and mCherry sorted transduced cells were subjected to gamma irradiation (IR) at different doses: 0.25 Gy, 0.5 Gy, 1 Gy, 2.5 Gy and 5 Gy. IR was delivered with a J L Shepherd Mark I Model 68 A Cesium 137 irradiator. 100–200 treated cells were plated in 6 well plates, and allowed to grow. Cells that retained viability despite IR formed colonies, and after 10–14 days, colonies were stained with Crystal Violet Solution (Sigma) following the manufacturer’s instructions and counted.

Antibodies against Mre11 (NBP2-59677) and Rad50 (NBP2-20054) were purchased from Novus Biologicals. Antibody against Nbs1 was described previously (28 (link)). DNA-PK inhibitor (NU7441) was purchased from Selleck Chemicals.