Genetic diagram of Bad-mediated radiosensitization of zebrafish neural tissue.

<p>IR activates the pro-apoptotic activity of Bad in a pathway that is either downstream of or parallel to p53 (dotted lines indicate that it is unclear whether this step occurs in a p53-dependent or –independent manner). Bad and Puma are dependent upon each other to promote IR-induced apoptosis. However, based on Bad’s established role as a sensitizer BH3-only protein and Puma’s reported role as an activator BH3-only protein, Bad likely functions upstream of Puma to induce IR-mediated apoptosis through the mitochondrial pathway.</p

Bad is required for IR-induced apoptosis in zebrafish embryonic neural tissue.

<p>(A) Shown are lateral views of 27-hpf embryos (head is top left in each panel) either uninjected or injected with 200 nmol of <i>bad ATG</i>, <i>bad e2i2</i> or mismatch (mm) MO. Half of each group of embryos were exposed to 15 Gy IR, and all were analyzed by the Casp3 assay. In control embryos (no inj and mm), IR-induced apoptosis occurs predominantly in the brain and all along the spinal cord (white arrowheads), whereas in <i>bad</i>-deficient embryos (ATG and e2i2), residual apoptosis is only observed in the head (arrowheads). (B) Fluorescence intensity, reflecting level of Caspase 3 activity, was measured in the spinal cords of at least 10 embryos from each group in (A) as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088151#pone.0088151-Sorrells1" target="_blank">[34]</a>. The fluorescence intensity in irradiated mismatch-MO-injected embryos was normalized to 1. (C) One-cell stage zebrafish embryos were injected with 100 nmol of <i>bad ATG</i>, <i>bad e2i2</i> or mm MO as indicated (“++” indicates that 200 nmol was injected to keep total concentration of MO constant between experimental groups) and irradiated and analyzed as in (A-B). Data represent one experiment, and the experiment was independently performed three times with similar results.</p

Bad does not augment p53 transcriptional activity.

<p>p53 transcriptional activity was analyzed in embryos injected with 50<i>mcherry</i> (cntl) or <i>hBAD</i>. Embryos were exposed to 8 Gy (or not) at 24 hpf. RNA was harvested from each group at 27 hpf and analyzed for gene expression changes by qPCR. Expression of the <i>gapdh</i> gene was measured to normalize <i>puma</i> and <i>p21</i> mRNA levels. All data was compared to unirradiated wild-type control-mRNA-injected data, which was adjusted to a value of 1. Control-injected <i>p53</i> mutant embryos irradiated at 24 hpf and harvested at 27 hpf were included as a negative control for p53-mediated transcriptional induction. Data represent one experiment, but the experiment was independently performed three times with similar results.</p

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

<p>(A) Shown are lateral views of representative tails from 27-hpf wild-type or <i>p53</i> mutant embryos injected with 50 pg of <i>mcherry</i> (cntl) or <i>hBAD</i> 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 <i>hBAD</i> mRNA was injected into wild-type (arrowheads), but not mutant, <i>p53</i> 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 <i>p53</i> mutant embryos were injected with 50 pg of mRNA encoding either <i>mcherry</i> control (cntl) or the constitutively active mutant <i>zbad 2SA</i>. At 8 hpf, embryos were analyzed for survival (defined by a beating heart) as performed previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088151#pone.0088151-Sorrells1" target="_blank">[34]</a>. 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 <i>bad</i> or the apoptotically-inactive <i>zbad bh3 mut</i>. 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 <i>p53</i> wild-type (left) or mutant (right) embryos after injection with 25 pg of mRNA encoding either <i>mcherry</i> (cntl), <i>zbad</i>, or <i>zbad bh3 mut</i>. 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 <i>p53</i>-independent manner (arrowheads).</p

Puma is required for Bad-mediated radiosensitization.

<p>(A) Shown are lateral views of representative tails from 27-hpf embryos exposed to 8 Gy IR after injection at the one-cell stage with 50 pg of mRNA encoding <i>mcherry</i> (cntl) or <i>hBAD</i> in addition to either 100 nmol of <i>puma</i> MO or mismatch (mm) control MO. Analysis of Caspase 3 activity shows that <i>hBAD</i>-mediated radiosensitivity (arrowheads) is dependent on <i>puma</i> expression. (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.</p