Fig. 2

p53 is required for Bad-mediated sensitivity to IR but not wortmannin.

(A) Shown are lateral views of representative tails from 27-hpf wild-type or p53 mutant embryos injected with 50 pg of mcherry (cntl) or hBAD mRNA. Embryos were exposed (or not) to 8 Gy IR at 24 hpf and analyzed three hours later by the Casp3 assay. Apoptosis was observed in the spinal cord after hBAD mRNA was injected into wild-type (arrowheads), but not mutant, p53 embryos. (B) Fluorescence intensity was measured in the spinal cords of at least 10 embryos from each group in (A). Data represent one experiment, but the experiment was independently performed three times with similar results. (C) One-cell stage wild-type or p53 mutant embryos were injected with 50 pg of mRNA encoding either mcherry control (cntl) or the constitutively active mutant zbad 2SA. At 8 hpf, embryos were analyzed for survival (defined by a beating heart) as performed previously [34]. Data represent one experiment, but the experiment was independently performed three times with similar results. (D) One-cell stage wild-type embryos were injected with 50 pg of mRNA encoding either zebrafish bad or the apoptotically-inactive zbad bh3 mut. At 8 hpf, embryos were treated with increasing concentrations of wortmannin, or DMSO vehicle alone. At 48 hpf, embryos were examined for survival. At least 10 embryos were analyzed per group in three independent experiments. E) Shown are lateral views of tails from p53 wild-type (left) or mutant (right) embryos after injection with 25 pg of mRNA encoding either mcherry (cntl), zbad, or zbad bh3 mut. Embryos were split into two groups and treated with either 0.3 μM wortmannin or DMSO vehicle beginning at 8 hpf and analyzed at 24 hpf by the Casp3 assay. Wild-type Bad synergizes with wortmannin to induce apoptosis in multiple tissues in a p53-independent manner (arrowheads).