Fig. 5: The ZO-1/YB-1 interaction regulates SG formation.

a Effect of arsenite (50 µM; 6 h) on YB-1, YB-3 and ZO-1 protein levels in BAECs determined by immunoblot analysis. Histogram referring to the quantification of protein levels relative to β-actin. (YB-1 n = 5; YB-3 n = 5; ZO-1 n = 3 independent experiments). Unpaired two-tailed Student’s t test. Arsenite treatment decreases the interaction between YB-1 and ZO-1. ZO-1 (b) or YB-1 (c) were immunoprecipitated (IP) from lysates of BAECs treated or not with arsenite (500 µM; 30 min). Non-immune IgG serves as control for non-specific co-immunoprecipitation. Levels of immunoprecipitated proteins were determined by immunoblot using the indicated antibodies. Histograms show the quantification of the ratio of YB-1 levels relative to ZO-1 (b, n = 5 independent experiments) or of ZO-1 levels relative to YB-1 (c, n = 3 independent experiments) present in the immunoprecipitates. Unpaired two-tailed Student’s t test. d GST pulldown assay of YB-1 using ZO-1 fragments (aa. 1-510 and aa. 511-1100) fused to GST (schematic representation of domain organizations of ZO-1 is shown in Supplemental Fig. S4c). Lysates of myc-YB-1 expressing BAECs were incubated with ~50 pM of either GST alone, GST-ZO-1(1-510) or GST-ZO-1(511-1100) prebound to glutathione agarose beads. Association of myc-YB-1 was revealed by anti-myc immunoblotting. Input shows the quantity of protein used for GST pulldown. e Immunofluorescence staining images show the colocalization (yellow) of YB-1 (green) and ZO-1 (red), using antibodies against YB-1 or ZO-1, in HUVECs in the presence or absence of arsenite (500 µM; 30 min). The measure of colocation was determined by Pearson’s colocalization coefficient of YB-1/ZO-1 on cell junctions of HUVEC (CT n = 23; CT + arsenite n = 25 cell junctions per condition). Unpaired two-tailed Student’s t test. f Decreased SG formation in YB-1 depleted cells. Images of immunofluorescence staining, and its measurement as a histogram, of SGs using antibodies against G3BP1 or against YB-1 in siCT, siZO-1, siYB-1 and combination of siZO-1 plus siYB-1 transfected HUVECs in presence of arsenite (500 µM; 30 min). Nuclei are stained with DAPI. The percentage of cells with SGs was calculated in n = 10 fields of view per condition and at least 25 cells/field. One-way ANOVA followed by Bonferroni’s multiple comparison tests. The downregulation efficiency of siRNA was confirmed by immunoblot. g Effect of overexpression of YB-1, ZO-1 or both on SG formation in ECs. BAECs expressing mycYB-1, mCherryZO-1 or both were treated with arsenite (500 µM; 30 min) and the percentage of cells with SGs of n = 10 fields of view per condition and at least five transfected cells/field was quantified. One-way ANOVA followed by Bonferroni’s multiple comparison tests. h Effect of arsenite on YB-1 and G3BP1 association in siCT or siZO-1 transfected HUVECs. G3BP1 was immunoprecipitated (IP) from lysates of BAECs transfected with siCT or siZO-1 and treated or not with arsenite (500 µM; 30 min). Non-immune IgG serves as control for non-specific co-immunoprecipitation. Levels of immunoprecipitated proteins were determined by immunoblot using the indicated antibodies. Histogram showing the quantification of the ratio of YB-1 levels relative to immunoprecipitated G3BP1. Note that arsenite treatment increases G3BP1 levels in lysates (n = 4 independent experiments). Unpaired two-tailed Student’s t test. Data are presented as mean values ± SEM. Source data are provided in the Source Data file.