Fig. 4: ZO-1 depletion increases viability and protects ECs against cellular stress.

a ZO-1 depletion protects ECs against cell death induced by arsenite treatment. Cell viability, measured by Trypan blue exclusion, of HUVECs transfected with siRNA against ZO-1, VE-cadherin or β-catenin and treated or not with arsenite (25 µM; 12 h) (n = 4 independent experiments). b Downregulation of ZO-1 decreases caspase-3 cleavage. Immunoblot analysis of cleaved caspase-3 levels in siCT, siZO-1, siβ-catenin and siVE-cadherin transfected BAECs with siRNA and treated or not with arsenite (50 µM; 6 h). Histogram referring to the quantification of cleaved caspase-3 levels relative to β-actin (siVE-cadherin + arsenite n = 3; all other conditions n = 4). Efficacy of siRNA-mediated knockdowns is shown in Supplemental Fig. S3a. c Effect of VEGF treatment (40 ng/ml) on cell viability, measured by Trypan blue exclusion, of siCT- or siZO-1-transfected HUVECs in presence of sodium arsenite (25 µM; 12 h) (n = 3 independent experiments). d Effect of VEGF treatment (40 ng/ml; 1 h) on the induction of G3BP1-positive SGs by sodium arsenite (500 µM for 30 min) in siCT- or siZO-1-transfected HUVECs (n = 8 fields of view per condition and at least 25 cells/field). e, f, g Effects of arsenite treatment on migrating ECs. Immunofluorescence analysis of SG formation and of ZO-1 levels in siCT or siZO-1 transfected HUVECs treated with arsenite (500 µM) or with arsenite and subjected to a wound healing migration assay for 30 min. e Images of immunofluorescence staining of G3BP1 and ZO-1 of cells in 1st and 2nd rows (leading) and in 3rd to 7th rows (followers) relative to the edge of the wound (white line) are shown. Nuclei were stained with DAPI. f The percentage of cells with SGs was calculated in leading and follower migrating cells as shown in (e) (n = 10 fields of view per condition and at least 15 cells/field). g ZO-1 fluorescence intensity was measured in leading and follower migrating cells as shown in (e) and normalized in each condition to the average intensity of the control (n = 12 fields of view per condition and at least 12 cells/field). h, Images of wound healing migration assays of siCT- or siZO-1-transfected BAECs treated with sodium arsenite (10 µM) and/or VEGF (40 ng/ml). Images were taken immediately after wounding of the cell monolayer (t = 0 h) and after 16 h of migration (t = 16 h). i Quantification of the percentage of wound closure at 16 h of migration, normalized to the control, is shown (siCT, siCT + VEGF, siZO-1, siZO-1 + VEGF, siCT + VEGF, siZO-1 + VEGF n = 9 migration areas per condition; siCT + VEGF + arsenite, siZO-1 + VEGF + arsenite n = 5 migration areas per condition). a–i Groups were compared using one-way ANOVA followed by Bonferroni’s multiple comparison tests; ns not significant. Data are presented as mean values ± SEM. Source data are provided in the Source Data file.