p53 protein is degraded by the proteasome and rescued by insulin in RP mutants.

<p><b>A)</b> Western blot analysis of p53 levels in 2 dpf wild type or <i>rpS7</i> mutant cells untreated or exposed to 20μM of the proteasome inhibitor MG132. <b>B)</b> Western blot analysis of p53 protein levels in either wild type or <i>rpS7</i> mutants exposed to 25 Gy ionizing radiation followed by the addition of 350nM insulin and/or 10mM Trolox. <b>C)</b> Model illustrating how impaired AKT activity by RP mutations may result in the failure of p53 to stabilize in response to ionizing radiation.</p

Early cell death of RP mutant cells requires p53.

<p><b>A)</b> Acridine orange staining allowing visualization of dying cells in wild type or <i>rpS7</i> mutants at 1 dpf that are uninjected, injected with the p53 MO, or injected with the missense MO. Size bar = 0.25mm. <b>B)</b> Quantification of (A). **<i>p</i><0.01. <b>C)</b> Acridine orange staining of a <i>rpS7</i> mutant is observed predominantly in the brain region (arrowhead) or distributed over the surface of the tail (white boxes). <b>D)</b> Scoring results from o-dianisidine staining allowing visualization of hemoglobin-expressing cells in clutches of either 111 embryos (+mis MO) or 177 embryos (+p53 MO) from an <i>rpS7</i> pairing.</p

Caspase 3/7 activation is increasingly impaired in RP mutants.

<p><b>A)</b> Caspase 2 and <b>B)</b> 3/7 activity is measured using a proluminescent caspase-3/7 DEVD-aminoluciferin substrate in 2 dpf embryos either untreated or exposed to 25 Gy ionizing radiation (IR). <b>C)</b> TUNEL analysis of 2 dpf wild type or <i>rpS7</i> embryos reveals cells with DNA fragmentation when embryos are exposed to 25 Gy IR. <b>D)</b> The number of TUNEL-positive cells in (<b>C</b>) are quantified. <b>E)</b> TUNEL-positive cells in both wild type and <i>rpS7</i> mutants are observed predominantly in the brain region (arrowhead) or in the tail tissue dorsal to the notochord (black box). *<i>p</i><0.05, **<i>p</i><0.01.</p

Lithium chloride restores p53 stabilization in RP mutant embryos and tumor cells.

<p><b>A)</b> Western blot analysis of p53 levels in 2 dpf wild type, <i>rpS7</i>, or <i>rpL11</i> mutant embryos either untreated or exposed to 25 Gy ionizing radiation followed by the addition of 100μM LiCl. Note the partial rescue of p53 stabilization in the RP mutant embryos when exposed to LiCl after IR. <b>B)</b> Quantification of the p53:actin ratio of the western blots from (<b>A</b>). <b>C)</b> Western blot analysis of p53 levels in <i>rpS7</i> MPNST tumor cells untreated or incubated with varying concentrations of LiCl. <b>D)</b> Western blot analysis of p53 levels in <i>p53</i>^{<i>M214K/M214K</i>} MPNST tumor cells untreated or incubated with varying concentrations of LiCl. * indicates either a p53-specific isoform or a degradation product.</p

p53 protein stabilization is impaired independently of <i>p53</i> mRNA levels.

<p><b>A)</b> qPCR analysis measuring levels of <i>p53</i> mRNA in wild type or <i>rpS7</i> mutants at 1 or 2 dpf either untreated or exposed to 25 Gy ionizing radiation. <b>B)</b> qPCR analysis of <i>p53</i> mRNA levels in wild type or <i>rpL11</i> mutants at 1 or 2 dpf either untreated or exposed to 25 Gy ionizing radiation. *<i>p</i><0.05. <b>C)</b> Western blot analysis of p53 protein levels and the quantification of the p53:actin ratio of in <i>rpS7</i> or <i>rpL11</i> mutants at 1 or 2 dpf either untreated or exposed to 25 Gy ionizing radiation. * indicates either a p53-specific isoform or a degradation product.</p