Azd7648

RPE-1 cells were seeded in T175 flasks and transfected with siRNAs the next day. One day after transfection, cells were arrested in mitosis with 100 ng ml⁻¹ nocodazole for 8 h. Mitotic cells were collected by mitotic-shake off, washed three times with culture medium and reseeded onto coverslips and T75 flasks. Cells growing on coverslips were fixed at 20 h after releasing from mitosis for analysis of chromosome 1 micronuclei by FISH. For dispersion analysis, after 20 h release from nocodazole, cells growing on T75 flasks were arrested in mitosis with Colcemid for 4 h. Mitotic cells were collected by mitotic-shake off, centrifuged onto glass slides using the Cytospin 4 cytocentrifuge and processed for FISH.
RPTECs were seeded in T75 flasks and transfected with Cas9 (TrueCut Cas9 protein v2, Thermo Fisher Scientific) in a complex with an sgRNA targeting chromosome 3p using Lipofectamine CRISPRMAX Cas9 transfection reagent (Thermo Fisher Scientific). During transfection, 3 μM of the DNA-PK inhibitor AZD7648 (MedChemExpress) was added for 24 h and washed out. Three days later, cells were arrested in mitosis with Colcemid for 6 h. Mitotic cells were collected by mitotic-shake off, centrifuged onto glass slides using the Cytospin 4 cytocentrifuge and processed for FISH.