Analysis of double strand break repair in MCF-7 monolayer and mammosphere populations. (a) Cells were exposed to 4 Gy 60Co γ-radiation and the relative degree of double-strand breakage (DSB) was determined by the comet assay under neutral conditions immediately after exposure and at the times indicated after exposure. (b) The 'comets' (n of about 100) were categorized according to the NIH LISTSERV (Comet Assay Interest Group web site) in which type 1 comets display the least DNA damage and type 5 the most. The error bars represent the mean ± standard error of the mean in both panels. The comets of the unirradiated cells are labeled Cont. (c) Expression of proteins involved in the NHEJ pathway of DSB repair in response to increasing doses of ionizing radiation. Lysates were prepared from unirradiated cells and from cells harvested one hour after exposure to 1 or 10-Gy 60Co γ-radiation and analyzed by immunoblotting with antibodies against several DSB repair proteins. Phospho-ATM and phospho-DNA-PKcs refer to phosphorylation of these proteins at Ser1981 and Ser2056, respectively. Actin served as a loading control.