Fig 2: AMOT is a p73-downstream effector for the regulation of endothelial junctions and actin dynamics. A, B Confocal microscopy analysis of HUVEC after p73 knockdown and XAV939 treatment. Stabilization of endogenous AMOT levels (A, pink) by XAV939 treatment rescues the morphology of endothelial AJ as shown by VE-CADH staining (A, orange, yellow arrows; B, green) and the organization of F-ACTIN (B, red). Nuclei were counterstained with DAPI (blue). Scale bar: 20 µm. (A, low panel) Quantitative analysis of VE-cadherin cell junction morphology. Bars represent mean ± SEM (n = 30 pictures per condition across three independent experiments). p values indicate the statistical differences between +shp73 and VECTOR values (***p < 0.001) and between +shp73 untreated and XAV939-treated cells (###p < 0.001). There were no significant differences among the morphological types between CTR and VECTOR

Fig 3: p73 is necessary for endothelial barrier integrity in vitro and in vivo. A Permeability assays to fluorescent dextran molecules in HUVEC monolayers comparing non-interfered (CTR), scrambled control (SCR), and p73-knockdown cells by siRNA (sip73). Cells were nucleofected and subsequently plated on the insert well. After 96 h, each insert was transferred to a fresh well plate and 250 kDa FITC-Dextran was added and incubated for 20 min at RT. Fluorescence (485 nm excitation/535 nm emission) was quantified in a plate reader. TAp73 expression level was analyzed by qRT-PCR in each case (left graph). B ECIS analysis of the effect of p73KD (+shp73) by TEER (trans-endothelial electric resistance). The electric parameters were measured for 48 h after plating the cells, which had been transduced with the indicated lentivirus 3 days ahead. The 48-h time course of TEER and of the basolateral membrane resistance values (Rb) are plotted, and graphs with the statistical analysis at the 24 h-time point (pointed by an arrow) are displayed. Three independent experiments were performed. C Amot mRNA expression levels in WT and p73KO mice retina were analyzed by qRT-PCR. D, E Immunofluorescence analysis of P7 WT, p73KO, TAp73KO (E) and DNp73KO (E) mice wholemount retinas stained with IB4 (red), anti-VE-CADH (green) (D) and anti-TER119 (green) (E). Scale bar: 20 μm. D Representative confocal microscopy images of vascular networks and vascular leading edge in WT retina show linear intercellular junctions (white arrows) with unicellular segments lacking VE-CADH (white arrowhead). On the contrary, lack of p73 results in retina vessels with interrupted staining (yellow arrows), even in multicellular vessels (blue arrowheads), and in aggregations of IB4 + EC (blue dashed arrows) close to sprouts with very little VE-CADH (blue arrows). Retinas from at least 3 mice per genotype were analyzed. E Vessel integrity was assessed by quantifying the percentage of erythrocytes (TER119 + cells) inside or outside the IB4 + blood vessels (right panel). At least three mice per genotype were considered, and a minimum of 20 pictures were quantified for each genotype. Bars represent the mean percentage ± SEM. p value was calculated with respect to the WT littermates for each genotype. *p < 0.05 **p < 0.01, ***p < 0.001, ns: no significant

Fig 4: p73 regulation of AMOT impinges on its downstream functions and controls AMOT-mediated YAP sequestration during cell contact inhibition. A Non-infected control HUVEC (CTR), and cells transduced with a non-silencing empty vector (VECTOR) or with an shRNA targeting p73 (+shp73) were seeded at 7.5 × 104 cells/cm2 and after 48–72 h, cells were treated with XAV939 for 24 h. Non-treated control cultures display frequent nuclear exclusion (yellow arrowheads) of YAP staining (red), whereas +shp73 cultures show a biased YAP distribution toward the nucleus (yellow arrows). Immunofluorescence analysis demonstrates that XAV939 treatment restores AMOT (green) levels and localization at intercellular junctions (white arrows) and correlated with a change in YAP localization in p73KD cells upon treatment (dashed yellow arrows). Orthogonal projections of the areas indicated by white dashed squares (1, 2, 3) are provided in Supplementary Fig. 2, showing that stabilized AMOT colocalizes with YAP at the plasma membrane as well as in the cytoplasm. (A, right graph) Quantification of YAP nuclei/cytoplasm ratio after p73KD ± XAV939 treatment. For these analyses, three independent infections were performed and at least five pictures from each experiment were randomly selected and quantified (n > 100 analyzed cells across the three independent experiments). The obtained data followed a logarithmic distribution; thus, for the statistical analysis, a log transformation was applied to the data. The computed P value is indicated in the graph. B, C Analysis of YAP (red) localization and proliferation (C, KI67, green) in HUVEC at different seeding density conditions (low—1 × 104 cells/cm2 versus high density—7.5 × 104 cells/cm2/High). Cell delimitation was discerned by β-CATENIN (B, green) staining. Nuclei were counterstained with DAPI (B, light blue). Scale bar: 20 µm. Under sparse culture conditions (low density), YAP localized mainly at the nucleus (yellow arrows), either in controls or in p73KD cells. Upon contact inhibition (high density) many CTR and VECTOR cells displayed YAP nuclear exclusion (white arrows); however, p73KD cells (+shp73) showed little or no nuclear exclusion (yellow arrows) upon confluency. Quantification of YAP nuclei/cytoplasm ratio was performed as in A (n > 60 cells for low-density and n > 100 for high-density conditions). C Quantification of the proliferation percentage after p73KD (right panel). Bars represent mean ± SEM of three independent experiments. ***p < 0.001, ns: no significant