Fig. 7

Knock down of SIRT6 abolished HNG’s protection on HG-induced endothelial senescence. (A) Western Blotting analysis (B) semi-quantification of SIRT6 in HUVECs transfected with SIRT6 or nontargeting siRNA. (C) Representative images of SA-β-gal staining and (D) quantification of SA-β-gal positive HUVECs treated with normal glucose (5.5 mM d-glucose, C), high glucose (15 mM d-glucose, HG), high glucose with [Gly14]-Humanin (15 mM d-glucose with 1 µM [Gly14]-Humanin, HG + HNG), including transfected with SIRT6 or nontargeting siRNA. Scale bar = 20 μm. (E–H) The mRNA levels of (E) P53, (F) P21 (G) P16, and (H) Cyclin D1 of the six groups measured by qRT-PCR. (I) Representative images of Western Blotting analysis and (J-N) semi‐quantification of (J) SIRT6, (K) p53, (L) p21, (M) p16, and (N) Cyclin D1 of the six groups. (O) The representative images of wound healing assay of HUVECs of the six groups. (P) The semi‐quantitative analysis of the distance of migration. Scale bar = 100 μm. (Q, R) Representative micrographs of EdU staining (Q) and quantification of EdU positive HUVECs. Scale bar = 100 μm. The values are expressed as the mean ± SEM. Experiments were carried out in triplicate and repeated three times. Results in B, E-H and P were analyzed with Student’s t-test. Results in D were analyzed with two-way ANOVA with Sidak’s multiple comparisons test. Results in J-N and R were analyzed with two-way ANOVA with Dunnett’s multiple comparisons test.