Fig. 4

High glucose downregulated the expression of SIRT6 to increase ROS production and endothelial senescence. (A) Representative images of Western blotting analysis and (B, C) semi-quantification of SIRT6 and SIRT1 in HUVECs treated with normal glucose (5.5 mM d-glucose, C), high mannitol (15 mM mannitol, M), high glucose (15 mM d-glucose, HG). (D, E) The mRNA levels of SIRT6 in HUVECs of C, M, HG, HG + NAC groups. (F) ROS detection of HUVECs treated with normal glucose (5.5 mM d-glucose, C), high glucose (15 mM d-glucose, HG), normal glucose with OSS-128,167 (5.5 mM d-glucose with 100 µM OSS-128167, C + OSS-128167), high glucose with OSS-128,167 (15 mM d-glucose with 100 µM OSS-128167, HG + OSS-128167) detected by flow cytometry. (G) Quantitative analysis of DCFH-DA fluorescence intensity in the four groups. (H) Representative images of SA-β-gal staining and (I) quantification of SA-β-gal positive HUVECs in the four groups. Scale bar = 20 μm. (J–L) The mRNA levels of (J) P53, (K) P16, (L) P21 of the four groups measured by qRT-PCR. (M) Representative images of Western Blotting analysis and (N–P) semi‐quantification of (N) p53, (O) p21 and (P) Cyclin D1 of the four groups. The values are expressed as the mean ± SEM. Experiments were carried out in triplicate and repeated at least three times. Results in B-E were analyzed with one-way ANOVA with Dunnett’s multiple comparisons test. Results in G, I-L and N-P were analyzed with two-way ANOVA with Sidak’s multiple comparisons test.