Fig. 7

topbp1^cas003 mutants manifest deficient DNA damage repair capability.

(A-H′) Immuno-staining of γH2AX (green), c-myb fluorescent in situ hybridization (red) and DAPI stain (blue) in sibling and topbp1^cas003 mutant embryos at 3.5dpf. (E′-H′) Magnified views of the regions in the dashed boxes showing γH2AX⁺; c-myb⁺; DAPI⁺ cell in the CHT region. Arrows indicate the γH2AX⁺; DAPI⁺ cells. The γH2AX⁺; DAPI⁺ cells are increased in topbp1^cas003 mutants comparing to siblings. Moreover, γH2AX⁺; c-myb⁺; DAPI⁺ cells can only be detected in topbp1^cas003 mutants (H′). Scale bars represent 50µm. (I) Immunoblotting showing increased γH2AX level in the CHT of topbp1^cas003 mutant embryos at 3dpf. (J) Procedure of the hydroxyurea (HU) recovery assay. Brown arrow line represents 250mM HU or mock treatment from 60hpf to 72hpf; green arrow line indicates removal of HU or mock treatment from 72hpf to 84hpf for recovery. (K-Q) The c-myb expression is further decreased in topbp1^cas003 mutant embryos under HU treatment. Wild-type siblings or topbp1^cas003 mutant embryos, after 12-hours mock treatment or after 12-hours 250mM HU treatment and sequential 12-hours recovery, were analyzed by either quantitative PCR (K) or c-myb WISH analysis (L-Q). c-myb level can be recovered in wild-type siblings, but not in topbp1^cas003 mutant embryos. The penetrance of the indicated phenotype is shown in the bottom left of each panel. Error bars represent SEM. * p<0.05; ** p<0.01; *** p<0.001. (L′-Q′) Enlarged views of the CHT region of embryos in the left columns.