Fig. 6. mTOR inhibition causes p53-dependent cell death by affecting Mdm2 protein levels in developing livers. (A) Analysis of Mdm2 (top) and mTOR (bottom) inhibition in E12.5 wild-type (+/+) and p53^-/- mutant livers. Panels show TUNEL-positive cells in representative liver sections treated with Nutlin-3 or rapamycin. Cell death for each genotype is shown (right) as a percentage increase in the number of apoptotic cells compared with non-injected mice (^*P<0.01; ^**P<0.001; Student's t-tests). Basal levels of TUNEL labeling were indistinguishable in non-injected wild-type and p53^-/- mutant embryos. Analysis on p53 mutants indicated that death of embryonic hepatocytes is also controlled by unknown p53-independent pathways. (B) Western blot analysis of total lysates of E12.5 livers of wild-type embryos treated or not with rapamycin. Rapamycin treatment causes a reduction in the levels of Mdm2 (top), which correlates with an increased phosphorylation of p53 on the S₁₈ residue (pS₁₈-p53; middle), whereas tubulin levels are unchanged. Levels of Mdm2 protein and p53 phosphorylation were quantified (right) after normalization with tubulin. ^*P<0.01; ^**P<0.001; Student's t-tests. (C) Immunohistochemical analysis of p53 phosphorylation on the S₁₈ residue of untreated (control), wild-type and p53^-/- mice injected with rapamycin. Quantitative analysis (right) is reported as the percentage increase of cells positive for p53 phosphorylation in rapamycin-injected wild-type mice over the number of positive cells in non-injected mice. ^*P<0.01; Student's t-tests. The numbers of embryos analyzed in these studies are indicated (n).