The <i>rs7</i>-mediated radiosensitizing phenotype is caused by an increase in <i>p53</i> mRNA expression.

<p>(A) Thirty hpf <i>rs7</i> siblings and mutants were identified based on morphology (similar to 1C). RNA was isolated, reverse transcribed using oligo-dT primers and analyzed for the expression of <i>p53</i> mRNA by qPCR. Expression of the <i>gapdh</i> gene was also analyzed to normalize <i>p53</i> mRNA levels. All data was then compared to sibling data, which was adjusted to a value of one. (B) <i>rs7</i> siblings and mutants were grown to 24 hpf and analyzed by whole-mount <i>in situ</i> hybridization with a probe complementary to <i>p53</i> mRNA. High levels of <i>p53</i> mRNA expression are evident in neural tissue (arrowheads) and the ICM (arrows). Pictures were taken first, and the embryos were subsequently genotyped to identify mutants and heterozygous or wild-type siblings. (C) RNA from (A) was reverse transcribed with random hexamers, and intron 9 of the <i>p53</i> gene was analyzed by qPCR to determine levels of <i>p53</i> pre-mRNA. Expression of <i>28S</i> RNA was also analyzed to normalize <i>p53</i> pre-mRNA levels. Similar results were obtained from an analysis of intron 4 (data not shown). All data was then compared to sibling data, which was adjusted to a value of one. (D) <i>rs7^+/−;p53^e7/e7</i> fish were incrossed to analyze <i>rs7</i> siblings and mutants in a <i>p53</i> homozygous mutant background. <i>Rs7</i> mutants and siblings were distinguished by morphology since loss of p53 does not prevent the <i>rs7</i>-mediated “curly-up” tail phenotype. RNA was harvested at 30 hpf and analyzed as in (C). (E) Protein was harvested from <i>rs7</i> siblings and mutants, and p53 and control morphants (injected at 400 µM) at 30 hpf and analyzed for p53 and Tubulin (as a loading control). ImageJ software was used to quantify band intensity from film. Shown below the blot are values for p53 divided by values for Tubulin (corresponding to above lanes) with <i>rs7</i> siblings normalized to one. (F) <i>rs7</i> sibling or mutant embryos were irradiated at 24 hpf with 8 Gy IR and analyzed 2 h later by whole-mount <i>in situ</i> hybridization with a probe complementary to <i>puma</i> mRNA. Arrowheads point to <i>puma</i> expression in neural tissue. (G) <i>rs7</i> sibling or mutant embryos in the <i>p53</i> wild-type or homozygous mutant background were irradiated at 24 hpf with 8 Gy IR and analyzed three hours later by immunofluorescence to detect activated Caspase-3. For panels (A), (C), and (D), error bars represent the standard error of the mean from at least three independent experiments. Panels (B) and (F–G) show representative data from at least three independent experiments. RT; reverse transcriptase. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen.1002922.s004" target="_blank">Figures S4</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen.1002922.s005" target="_blank">S5</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002922#pgen.1002922.s006" target="_blank">S6</a>.