Fig. 3

RP-deficient zebrafish show activation of the DNA damage checkpoint pathway. (A) Phosphorylation of residue Ser139 of histone H2A.X was induced in embryos that had been injected with an Rps19-specific morpholino (MO). Western blotting was performed at 24 hpf. Staining of tubulin was used for a loading control. Molecular masses are shown on the right. Wt, wild type. (B) Phosphorylation of residue Ser345 in Chk1 kinase was increased in Rps19-deficient embryos (MO). Western blotting was performed at 24 hpf. (C) Embryos that had been injected with an Rps19 morpholino had increased levels of p53; the treatment of morphants with 3 nM of Chk1 inhibitor PF477736 (PF), 10 nM of the ATR and ATM inhibitor CGK733 (GCK), or 3 nM of the ATM inhibitor KU60019 (KU) reduced p53 levels. Western blotting was performed at 24 hpf. (D) Treatment of Rpl11 mutants with inhibitors of the ATR-ATM-Chk1 pathway resulted in downregulation of the p53 targets p21 and puma. Gene expression was analyzed by using RT-qPCR. The fold change of gene expression was calculated relative to expression in wild-type siblings. Means±s.d. are shown. (E) Embryos that had been injected with a morpholino against Rps19 (Rps19 MO) had few red blood cells at 3.5 days post-fertilization. The treatment of Rps19 morphants with PF477736 partially rescued this hematopoietic defect.