Fig. 5

Humanin prevents HG-induced senescence. (A) Representative images of SA-β-gal staining and (B) quantification of SA-β-gal positive HUVECs treated with normal glucose (5.5 mM d-glucose, C), high mannitol (15 mM mannitol, M), high glucose (15 mM d-glucose, HG), high glucose with [Gly14]-Humanin (15 mM d-glucose with 1 µM [Gly14]-Humanin, HG + HNG), [Gly14]-Humanin (1 µM [Gly14]-Humanin, HNG). Scale bar = 20 μm. (C) ROS detection of HUVECs treated with C, M, HG, HG + HNG, HNG. (D) Quantitative analysis of DCFH-DA fluorescence intensity in the C, M, HG, HG + HNG, HNG group. (E–H) The mRNA levels of (E) P53, (F) P16, (G) P21, (H) SIRT6 of the five groups measured by qRT-PCR. (I) Representative images of Western Blotting analysis and (J-N) semi-quantification of (J) p53, (K) p21, (L) Cyclin D1, (M) SIRT1 and (N) SIRT6 of the five groups. (O) Cell proliferation of C, M, HG, HG + HNG, HNG groups assessed by CCK-8 assays. Optical density (OD) = 450 nm. (P) The representative images of wound healing assay of HUVECs of the five groups. (Q) The semi‐quantitative analysis of the distance of migration. Scale bar = 100 μm. (R, S) Representative micrographs of EdU staining (R) and quantification (S) 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 and D were analyzed with one-way ANOVA with Tukey’s multiple comparisons test. Results in E-H, J-N and S were analyzed with one-way ANOVA with Dunnett’s multiple comparisons test. Results in Q were analyzed with two-way ANOVA with Dunnett’s multiple comparisons test.