Fig. 2

AND-1 depleted DT40 cells display spontaneous accumulation of DNA double strand breaks (DSBs) and DNA damage checkpoint activation. a Cell cycle distribution of AND-1 depleted cells. and-1-aid cells were incubated with auxin for indicated times, stained with propidium iodide (PI), and DNA content was analyzed by flow cytometry. b and-1-aid cells were incubated with or without auxin for 12 h, caffeine was added, and further incubated for the indicated times. DNA content was analyzed by flow cytometry. c Total cell lysates were prepared from and-1-aid cells at the indicated time points and analyzed by Western blotting. d and-1-aid cells were incubated with or without Auxin for 12 h, caffeine and colcemid were added, and further incubated for 2 h. Metaphase spreads were prepared from and-1-aid cells. Examples of intact and damaged chromosomes (the chromosomes are identified by shape in DT40) are shown. Scale bars represent 10 μm in left panel, 1 μm in enlarged picture (chromosome 2). e and-1-aid cells were incubated with auxin for 8 h and γH2AX or RAD51 foci were visualized by immunostaining with specific antibodies. Scale bars represent 25 μm in upper panels, 5 μm in enlarged pictures. f DSB detection via PFGE. and-1-aid cells were incubated with auxin for the indicated times, collected into agarose plugs and their DNA was separated by size on an agarose gel. Under the electrophoresis conditions used, high molecular weight genomic DNA remains in the well, whereas lower molecular weight DNA fragments (several Mb to 500 kb) migrate into the gel and are compacted into a single band