Fig 2: DIAPH1-related gene network, KEGG pathway analysis, and GO enrichment analysis. (A) A total of 50 available experimentally determined DIAPH1-interacting proteins using STRING tool. (B) An intersection analysis of the DIAPH1-interacting and -correlated genes. (C) The correlation between DIAPH1 expression and four intersected genes expression in PAAD. *** p < 0.001, in Spearman’s rho. TPM, transcripts per million. (D) GO enrichment and KEGG pathway analyses based on the DIAPH1-interacting and -correlated genes. Adjusted p-values were obtained from multiple hypotheses test using the Benjamini–Hochberg procedure; p. adjust <0.05 was considered statistically significant. (E) Correlation between DIAPH1 and intersected four genes expression in pan-cancer, in purity-adjusted partial Spearman’s rho.

Fig 3: NAA80 preferentially binds monomeric actin in vitro and in cells.(A) Domain diagram of NAA80, showing the two major constructs used in this study. (B) In vitro acetylation by NAA80?N (lacking the 77–amino acid N-terminal extension, or ?N) of nonacetylated ß/?-actin analyzed by Western blotting, using acetylation- and isoform-specific anti–Ac-ß-actin antibody. Ac-ß/?-actin purified from WT cells and actin acetylated by full-length NAA80 (NAA80FL or FL) were used as controls. Loading was controlled by anti–pan-actin staining. (C) Rescue of acetylation in NAA80 KO cells by NAA80?N analyzed as in (B). Lysate from WT cells was used as a control. Anti–green fluorescent protein (GFP) staining confirmed NAA80?N expression in cells. (D) Western blot analysis of the presence of endogenous NAA80 in the soluble (G-actin) and pellet (F-actin) fractions of HAP1 WT cells using custom anti-NAA80 antibody (1:200). Controls are profilin, which binds only G-actin, and Dia1, which binds both G- and F-actin. NAA80 KO cells are shown as a control for custom antibody specificity. Left: Representative Western blots (fig. S8). Right: Quantifications, showing data points (red circles) and means ± SD (n = 5). The statistical significance of the data was determined using an unpaired two-sided Student’s t test (***P < 0.001). (E) Representative HeLa cells expressing GFP-NAA80FL (GFP-FL) or GFP-NAA80?N (GFP-?N) and stained with Alexa Fluor 546 phalloidin to visualize F-actin. Scale bar, 10 µm. Line scans illustrate the lack of NAA80 colocalization with F-actin, as also confirmed by whole-cell correlation coefficient analysis on n = 15 cells (fig. S2, D and E). (F) Cosedimentation of NAA80FL with F-actin (Ac-a-actin) ± tropomyosin (Tpm). Data points correspond to the mean from three independent experiments ± SEM (fig. S7A). The maximum molar binding ratio (Bmax) and observed dissociation constant (Kobs) were calculated using a nonlinear least square fit (see Materials and Methods). (G and H) Isothermal titration calorimetry (ITC) titrations of NAA80FL and NAA80?N (CoA) into Ac-a-actin (latrunculin B, or LatB), using the indicated experimental conditions. The dissociation constant (KD) and binding stoichiometry (N) were derived from fitting to a one-site binding isotherm (red line). Errors correspond to the SD of the fits. Open symbols correspond to control titrations into buffer.

Fig 4: Schematic diagram of signal transduction pathways involved in Ang II-induced endothelial hyperpermeability via AT1R/RAGE/mDia1/Src/β-catenin/VE-cadherin.a As Ang II binds to and stimulates AT1R, the expression and secretion of HMGB1 can be increased by NF-κB activation. NF-κB-mediated expression of proinflammatory molecules, including RAGE itself, can occur. HMGB1 binds to RAGE, which can induce RAGE-mediated activation of Src/β-catenin/VE-cadherin via mDia1. b Blockade of RAGE activation by sRAGE attenuates the Ang II-induced increase in endothelial hyperpermeability by inhibiting RAGE-mediated signaling pathways

Fig 5: Soluble RAGE (sRAGE) attenuates Ang II-induced endothelial hyperpermeability.a HUVECs were treated with sRAGE (2 μg/ml) for 1 h and were then subjected to Ang II treatment for 4 h. Endothelial permeability was assessed in media from the lower chambers after the addition of FITC-dextran 40 to the upper chambers for 1 h (n = 3 for each lane). b TEER was measured every 2 h. *p < 0.05 vs. control, #p < 0.05 vs. Ang II (n = 3 for each lane). c Immunocytochemistry of VE-cadherin (green) and DAPI (nuclei, blue) was performed, and samples were directly examined under a confocal microscope (white scale bars: 50 μm in merged images and 5 μm in magnified images; ×400 magnification). The main images were selected from representative regions. d HUVEC extracts were analyzed by western blotting to assess phospho-Src, phospho-β-catenin, and phospho-VE-cadherin expression. Expression values were normalized to those of Src, β-catenin, and VE-cadherin (n = 4 for each lane). e The protein levels of AT1R, RAGE, and mDia1 were determined by western blotting. Expression was normalized to that of GAPDH (n = 3 for each lane). f HMGB1 release in supernatants was measured by ELISA and western blotting (n = 3 for each lane). The values are presented as the means ± SEMs. *p < 0.05; **p < 0.01; ***p < 0.001; ns not significant by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test