Fig. 1: H/R increased endothelial permeability and induced extracellular secretion of HMGB1 in HBMVECs. | Cell Death & Disease

Fig. 1: H/R increased endothelial permeability and induced extracellular secretion of HMGB1 in HBMVECs.

From: Sirtuin 1-dependent regulation of high mobility box 1 in hypoxia–reoxygenated brain microvascular endothelial cells: roles in neuronal amyloidogenesis

Fig. 1

a, b HBMVECs were exposed to hypoxia/glucose deprivation for 1 h followed by reoxygenation (H/R) in complete medium for the indicated times. The infiltrated FITC-dextran level in the abluminal side (a n = 6) and the expression levels of the junctional proteins VE-cadherin (n = 6), ZO-1 (n = 6), Occludin (n = 3) and Claudin-5 (n = 3) (b) during H/R for 6 h were measured; α-tubulin or GAPDH was used as a loading control. Representative blots are presented, and each graphs indicate the change in expression level relative to the level at 0 hr of normoxic control (NC) group, and vertical bars indicate mean ± SD. c Expression levels of VE-cadherin (n = 5), ZO-1 (n = 5), ICAM1 (n = 5), Occludin (n = 6) and Caludin-5 (n = 6) in HBMVECs with 24-h H/R were analysed by western blot analysis. β-Actin was used as a loading control. Representative blots are presented. Each bar graphs indicate the relative expression level of each protein to that of NC (mean ± SD; *p < 0.05 versus NC by unpaired t-test). d Cell viability of HBMVECs exposed to 24-h H/R was measured by MTT assay. Bar graphs indicate the mean ± SD. ***p < 0.001 versus NC. e Levels of HMGB1 protein in the nucleus and cytosol were analysed by western blotting. Lamin B and α-tubulin were used as loading controls for nuclear and cytosolic proteins, respectively. f HBMVECs with or without H/R treatment were fixed in paraformaldehyde and stained with an anti-HMGB1 antibody. Arrows indicate the cytosolic HMGB1 in cells treated with H/R for 2 h; not observed in NC. Horizontal white bars indicates 50 μm. g Levels of extracellular HMGB1 protein in CM from HBMVECs with or without 24-h H/R exposure were measured by western blotting; β-actin was used as a loading control (n = 4-5, mean ± SD; *p < 0.05 versus NC by unpaired t-test). Representative blots are presented.

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