Assessment of cellular radiosensitivity in the colony formation assay. A. Cellular radiosensitivity of p.R215W mutant cells (HCC1395) compared with wild type breast epithelial cells (MCF10A) as measured by the colony formation assay after irradiation at doses of 0, 0.1, 0.25, 0.5 or 1Gy. The surviving fraction is presented as the mean value with SEM from at least 3 independent experiments. B. Induction of PARP1 cleavage after irradiation with 2 Gy in p.R215W mutant cells (HCC1395) compared with wild type breast epithelial cells (MCF10A) as measured by immunoblotting of cleaved PARP1 (89 kDa) and total PARP1 (116 kDa). C. Immunoblot analysis of radiation-induced ATM signalling in HCC1395 cells compared with MCF10A. Cells were untreated or irradiated with 0.5, 1, 2, 4 or 6 Gy as indicated. Protein extracts were prepared 30 min after irradiation and were analysed through Western blotting for their immunoreactivity towards the phosphorylated forms of SMC1 (pSer966, top panel), KAP1 (p824, middle panel), and CHEK2 (pSer19, bottom panel). β-actin served as the loading control in each experiment.