Fig 2: The ß-catenin C-terminal interactions are required for repression of p53 activity.(a) Schematic of wild-type (WT) and mutant ß-catenin proteins. (b,c) p53 activity and Gal4BD-p53 activity, respectively, in WT mouse aortic SMCs electroporated with the indicated expression vectors. *P<0.05; **P<0.01; ***P<0.001; comparisons done by one-way ANOVA and Dunnett's multiple comparisons test. In b, n=3; in c, n=6. Luminescence (Lum) was normalized to ß-galactosidase activity (ß-gal) to control for transfection efficiency. (d) Immunostaining of mouse aortic SMCs in confluent and subconfluent monolayers for ß-catenin and CBP. Nuclear staining by DAPI. Scale bar, 20 µm. (e) Western blot analysis of indicated proteins in cytosolic and nuclear fractions of WT mouse aortic SMCs. (f) Evaluation of acetylated p53 by sandwich ELISA in WT mouse aortic SMCs treated with vehicle (DMSO) or 50 µM ICG001, an inhibitor of ß-catenin/CBP interaction. Levels of acetylated p53 were measured by spectrophotometric determination of absorbance at 450 nm, normalized to input and expressed as fold change of control cells. ****P<0.0001 by two-tailed t-test. n=3. (g) Left: western blot analysis of indicated proteins in mouse aortic SMCs with annotated treatments. Right: densitometric analysis of normalized CBP protein levels in the indicated conditions. ****P<0.0001 by two-tailed t-test. n=3. (h) Evaluation of acetylated p53 as in f, in mouse aortic SMCs with indicated treatments. **P<0.01; ***P<0.001; comparisons done by one-way ANOVA and Dunnett's multiple comparisons test. n=3. In b,c,f–h, data represent the mean ±s.e.m. A, alanine residue in position 164; C, conserved helix C; CTD, carboxy-terminal domain; D, aspartate residue at position 164 on the first armadillo repeat; NABP, nucleic-acid-binding protein subset (soluble nuclear fraction); NS, not significant; NTD, amino-terminal domain. In a numbered ovals are armadillo repeats, and critical regions and respective interacting proteins are indicated by brackets.

Fig 3: Loss of p53 suppresses the vascular phenotype caused by loss of ß-catenin.(a) Left: immunostaining for Bax, a known p53 target gene, and a-SMA at E11.5. Scale bar, 20 µm (PDA); 50 µm (FDA). Right: quantification of Bax expression in the media of aortas, expressed as percentage of medial area positive for Bax. Data represent the mean ±s.e.m. *P<0.05 by two-tailed t-test. n=4. (b,e) E12.5 embryos of the indicated genotypes. Scale bar, 1 mm. (c) Percentage of E12.5 embryos of the indicated genotypes in three phenotypic categories: normal, early resorption or advanced resorption. **P<0.01; ****P<0.0001; comparisons done by ?2-test. (d) H&E-stained sections of FDAs of the indicated genotypes. Scale bar, 50 µm.

Fig 4: Changes in Cell Cycle genes due to ethanol.Overall, ethanol exposure altered more genes in this KEGG pathway in WT mice than p53 KO mice. Several of the genes that were increased in WT mice after ethanol were decreased in p53 KO, and vice versa. Same convention as Fig 3.

Fig 5: Genes with significant interactions between Ethanol Treatment and p53 Genotype Expression in subregions of the somatosensory cortex.Note that each of these 3 genes are involved in neuronal-related functions (cell adhesion or neurite extension. Note that for two of the genes (Ephb1, Ctgf) the direction of change in two different layers was an increase following ethanol in the p53 KO mice but a decrease following ethanol in the WT mice. The opposite pattern of change was seen in Layer 6 for Camk1 n = 6.