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. 2016 Sep 11;9:306–319. doi: 10.1016/j.redox.2016.09.003

Fig. 1.

Fig. 1

Protein S-glutathionylation in endothelial cells is increased under conditions of diabetes and hypercholesteremia. Glutathionylated proteins (PrS-SG) is elevated in diabetic endothelial cells (ECs). A: Representative images of immunofluorescence staining of PrS-SG in ECs isolated from diabetic and non-diabetic control subjects. B: The intensity of PrS-SG staining in ECs from diabetic subjects (n=5) and control subjects (n=5) was quantified. From each subject sample, 20–30cells were immunostained for PrS-SG, and the fluorescence density was measured and averaged. Data represent mean±SEM. *p≤0.05. C: PrS-SG is elevated in aortic ECs in hypercholesterolemic mice. Representative images showing the localization and levels of PrS-SG in the cross sections of aortae from ApoE-/- mice fed a normal chow (ND) and a Western diet (WD) for two weeks. Freshly prepared aortic tissue cross sections were incubated with 10 mM NEM to alkylate free thiols, fixed with ice-cold acetone, and immunostained for PrS-SG (red channel), CD31(EC marker, green channel) and α-actin (smooth muscle cell marker, green channel). The first and the third row shows high magnification views of yellow-boxed areas. Scale bars are 50 µm. D: Two-week WD feeding dramatically increased the plasma levels of cholesterol, but not triglycerides in AopE-/- mice. Data represent mean±SEM (ND: n=7; WD: n=12). *p<0.05, compared with ND group. E: PrS-SG is elevated in cultured HAECs exposed to diabetic conditions. HAECs were treated with vehicle (50 μM BSA, 5 mM D-glucose, 20 mM mannitol) or high levels of palmitate-BSA conjugate and high glucose (HPHG) at indicated concentrations for 24 h. Cells were then incubated with 10 mM NEM for 15 min to alkylate free protein thiols, and the cleared cell lysates underwent Western blotting analysis with the anti-GSH antibody under nonreducing condition. The right panel is the image with short exposure. The β-actin blot serves as a loading control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)