Fig 2: Maturation of leaflets and commissures. (A) Immunolocalization at P6 of Nfatc1 (green) and Prox1 (red) in VV of WT mice administered control solvent or cyclosporin (n = 5 versus 8 VVs, P < 0.05, ?2). (B) Immunolocalization of Foxc2 (green) and Prox1 (red) in VVs of mice of the indicated genotypes. n = 6 per condition. (C and D) Quantification of leaflet length and stage of VV development in mice of the indicated genotypes. Error bars represent means ± SEM. t test for leaflet length. (C, D, G, H, and I) Colors represent developmental stage (see key). The number of VVs analyzed for each condition is given above each bar. *, P < 0.05; **, P < 0.005 (?2 for developmental stage). (E) Immunolocalization of Cx37 (arrowheads) and Cx43 in VV leaflets at P6. n = 4. (F) Localization of Prox1, Foxc2, and SMA at P6 in Gja4-/- vein. n = 6 (G and H) Stages of VV development reached in mice of the indicated genotypes. (I) Localization of Prox1, GFP, and SMA in Gjc2GFP/GFP KO reporter vein at P2 (n = 4), and stages of VV development reached in mice of the indicated genotypes. (J) Localization of Cx47-expressing VV cells (arrowheads) of Gjc2GFP/+ reporter mice at P6. n = 4. Multichannel images are reproduced in supplementary data. Bars, 20 µm. (A–J) Blue stain is PECAM1.

Fig 3: GJA1 mRNA/protein expression is correlated with AD pathogenesis, dementia and known AD risk factor genes. GJA1 mRNA expression is correlated with the MMSE score in the prefrontal cortex region in the ROSMAP cohort (a), the NTrSum score in the cortex region frontal pole (BM10) the MSBB cohort (b), the Braak score in the parahippocampal gyrus (BM36) in the MSBB cohort (c), and the CDR score in BM36 in the MSBB cohort (d), and CERAD in BM36 in the MSBB cohort (e). The GJA1 protein level is also correlated with the plaque mean density (PLQ_Mn) in the frontal pole (BM10) in the MSBB cohort (f). Inset was the correlation coefficient along its p value between individual clinical traits and GJA1 mRNA/protein level. Severity of AD symptoms was classified based on each individual AD neuropathological and cognitive traits according to the criteria established in the ROSMAP clinic codebook [91, 92]. g. Gja1 mRNA expression is correlated with a vast majority of the ADGWAS genes in the MSBB (BM10, BM22, BM36 and BM44) and ROSMAP RNA-seq datasets. Colors represent Correlation coefficients: red, high; blue, low; yellow, in between. * and ** stand for significance at 5 and 0.5% level, respectively while ns indicates insignificant correlation and na stands for “not applicable”. Shown in the heatmap are only the ADGWAS genes that passed RNA-seq data preprocessing, normalization and annotation

Fig 4: Immunofluorescence detection of Cx26, Cx32, Cx43, Cx46 (Alexa 546, red) and Ki67 (Alexa 488, green) in invasive ductal breast cancers prior to (pre-) and after (post-) neoadjuvant chemotherapy. Cell nuclei were stained using Hoescht (blue). Punctuate cell membrane associated and cytoplasmic Cx26 (1st row) and Cx32 (2nd row) levels and the proliferating (green) tumour cell fractions were reduced after neoadjuvant therapy in cases originally scoring 3+. Cx43 (3rd row) and Cx46 (4th row) levels did not change after neoadjuvant chemotherapy in cases of high scores (3+). Pre-chemotherapy Cx43 and post-chemotherapy Cx46 samples are not stained for Ki67.

Fig 5: Evaluating the purity of primary cultured astrocytes and Cx43 expression in astrocytic subcellular components. Primary astrocytes were prepared from newborn mice. a Western blotting analyses of Cx43 levels were conducted with samples prepared from astrocytes from the control, OGD/R, OGD/R-SalB, and OGD/R-CBX groups. Representative protein bands are displayed for total protein extractions and the plasma membrane and cytoplasm compartments. b After OGD/R injury, Cx43 levels were significantly upregulated in the cytoplasm but downregulated in the plasma membrane. SalB treatment reversed these effects. The four groups did not significantly differ in total Cx43 levels. We evaluated the statistical significance with ANOVA and Duncan’s multiple comparisons test. *p < 0.05, **p < 0.01, and ***p < 0.001. Scale bar = 50 µm