Fig. 5. Evidence for DNA damage in Arabidopsis csn mutants. (A) Accumulation of the phosphorylated H2AX variant H2AX in a histone preparation (13 µg) from 2-day-old (same size) and 7-day-old (same age) wild-type seedlings and 7-day-old csn3, csn4 and csn5ab mutant seedlings as detected with a H2AX-specific antibody (Friesner et al., 2005). A Coomassie-stained band serves as a loading control. H2AX transcription was not altered between the wild type and the csn mutants (data not shown). (B,C) Immunostaining of wild-type and csn4 mutant root tip cells with the anti-H2AX antibody (green) identifies subnuclear H2AX-specific foci that may mark sites of damaged DNA in csn4 mutants (C, left panel). The samples were counterstained using the DNA stain DAPI. Scale bars: 5 µm. (D) Activation of the stress-induced WEE1:GUS reporter construct in wild-type seedlings, bleomycin-treated (12 hours, 5 µg/ml) wild-type seedlings and in untreated csn mutants. Scale bars: 2 mm in wild type; 0.5 mm in csn3, csn4 and csn5ab. (E-M) Confocal images of primary roots of 7-day-old dark-grown wild type and csn mutants following the TUNEL assay (left column). Roots were counterstained with DAPI (right column). For the positive control, fixed root material was subjected to treatment with DNase I. For the negative control, terminal transferase was omitted from the TUNEL reaction. The experiment was repeated three times and a representative image from one experiment is shown. Scale bars: 1 mm in wild type, csn5a and csn5b; 0.5 mm in csn3, csn4 and csn5ab. (N) Confocal images of a 7-day-old bleomycin-treated (12 hours, 5 µg/ml) dark-grown wild-type seedling following the TUNEL assay (left panel). Roots were counterstained with DAPI (right panel). Scale bar: 1 mm.