Fig. 1: PNUTS is an aging-regulated protein and regulates senescence in endothelial cells.

a HUVECs were cultured for 3 or 17 passages (P) and PNUTS expression was measured by qRT-PCR (n = 4). b Aortic intima was isolated from young (8–12 weeks old) and aged (18–20 months old) mice and immediately lysed for RNA isolation. Intima RNA was sequenced. c–f HUVECs were transfected with siRNAs targeting PNUTS or a control sequence. c PNUTS protein levels were determined by WB at 48 h after transfection, relative to alpha-tubulin. Densitometric quantification is depicted on the right (n = 7). d Changes in p21 expression were assessed by qRT-PCR. Expression values are relative to siControl-treated HUVECs and normalized to RPSA mRNA (n = 6). e The percentage of senescent HUVECs was analyzed by staining for senescence-associated β-Galactosidase (SA-(β-Gal) 72 h after transfection. Images were taken (4 fields per sample) and the percentage of senescent cells (blue) over the total number of cells in each field was calculated in n = 3 independent experiments, 2–3 biological replicates per group and experiment). f Cell proliferation was assayed by testing the incorporation of EdU after transfection (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001. Error bars depict the standard error of the mean (SEM).