Effect of the HG-induced “memory” on cell growth and aging in HUVECs.

HUVECs were cultured in normal glucose media (D-glucose, 5 mM) for 6 days (NG), high glucose media (30 mM) for 6 days (HG), 3 days of normal glucose supplemented with D-mannitol (25 mM) followed by another 3 days of normal glucose media (MN), or 3 days of high glucose followed by another 3 days of normal glucose media (HN). (A) Cells were fixed and stained for SA β-Gal activity. Histogram represent the percentage of SA β-Gal positive cells (n = 5 image per group). (B) Cell count per microscopic field at different time points during cell subculture. (n = 3 image per group) (C) Relative ROS production. (D-E) Immunoblotting and quantification of SIRT1 expression. Values were normalized to GAPDH. (F) SIRT1 deacetylase activtity. (G-K) Immunoblotting and quantification of p300, SIRT1, p21, and Ac-p53 protein expression. For p300, SIRT1, and p21, values were normalized to β-actin; for Ac-p53, normalized to total p53. Value was normalized to total p53. *P < 0.05.