Supplementary MaterialsSupplementary Figures. of forkhead box M1 (FOXM1), a critical transcription factor for cell cycle progression and senescence. Overexpression of FOXM1 ameliorates SIRT6 deficiency-induced endothelial cell senescence. KL1333 In this work, we demonstrate the role of SIRT6 as an anti-aging factor in the vasculature. These data may provide the basis for future Rabbit polyclonal to ZBED5 novel therapeutic methods against age-related vascular disorders. siRNA. knockdown with siRNA treatment was confirmed by western blot analysis (Physique 2A). SIRT1 and SIRT6 downregulation significantly increased the population of SA -gal-positive cells 6 d after siRNA treatment, but knockdown did not induce endothelial senescence (Physique 2B, ?,2C).2C). The number of SA -gal positive cells in knockdown cells was 2.6-fold higher than that in knockdown cells. We confirmed knockdown-induced senescence using a different sequence of SIRT6 siRNA (siSIRT6*, Supplementary Physique 1AC1D). These data suggest that the downregulation of SIRT6 expression itself is enough to induce endothelial cell senescence. Open in a separate window Physique 1 SIRT6 expression is usually inhibited in endothelial cells during oxidative stress-induced or replicative senescence. (A) Representative image of SA -gal-positive HUVECs 10 d after the addition of H2O2 (200 M). (B) The percentage of SA -gal-positive senescent HUVECs that were treated with 200 M H2O2 for 1 h and then cultured for the indicated time to generate oxidative stress-induced senescence. The data represent the mean percentage SD (n = 3). * 0.01 vs. control. (C) Western blot images to analyze the expression of SIRT1, SIRT2, SIRT3, SIRT5, and SIRT6 in HUVECs at 1, 3, 5, or 10 d after addition of KL1333 H2O2 (200 M). (D) SA -gal staining images for young (PDL8) and aged (PDL36) cells. (E) The percentage of SA -gal-positive HUVECs that were passaged to induce replicative senescence. The data are shown as the mean SD (n = 3). * 0.01 vs. young cells. (F) The expression of SIRTs in young and aged HUVECs. An antibody realizing -actin was used as a loading control. Open in a separate window Physique 2 Knockdown of SIRT6 expression induces endothelial cell senescence. (A) Western blot analysis showing the KL1333 knockdown expression of SIRT1, SIRT3, and SIRT6 in HUVECs treated with siRNAs, respectively. Total protein was extracted from cells 1 and 3 d after siRNA treatment. (B) The representative images obtained from SA -gal-stained HUVECs. The cells transfected with the indicated siRNA (25 nM) were re-transfected with the siRNA 3 d after the first siRNA treatment. After 6 d from your first transfection, cells were stained for SA -gal. (C) The percentage of SA -gal-positive senescent cells at 6 d after siRNA transfection. The data are shown as the mean SD (n = 3). * 0.05 vs. control siRNA. SIRT6 is usually involved in the maintenance of endothelial cell function Senescent endothelial cells have impaired angiogenic function and are susceptible to inflammatory responses. To evaluate the effect of knockdown on capillary tube formation and inflammation in HUVECs, cells were transfected with 25 nM control, siRNA. When endothelial cells were cultured on Matrigel, the cells created capillary-like tube network. and siRNA-transfected HUVECs on Matrigel showed reduced branch points and very short tubes (Physique 3A). Moreover, knockdown inhibited eNOS and KLF2 expression (Physique 3B), which play essential roles in maintaining endothelial integrity [18, 19]. Depletion of SIRT6 resulted in an increase in the inflammatory responses of endothelial cells (Physique 3CC3E). knockdown increased ICAM-1 expression but not E- and P-selectin expression. TNF–treated HUVECs highly expressed ICAM-1 and E-selectin. Interestingly, siRNA treatment upregulated TNF–induced ICAM-1 and E-selectin expression compared to control siRNA treatment with TNF-. Open in a separate window Physique 3 Downregulated expression of SIRT6 induces endothelial cell dysfunction. (A) Effect of siRNA on tube formation in HUVECs. HUVECs transfected with 25 nM of the indicated siRNA were cultured on Matrigel to check angiogenesis activity of endothelial cells. The representative micrographs of tube formation in HUVECs. (B) Western blot analysis showing the result of siRNA for the manifestation of eNOS and KLF2. -Actin was utilized as a launching control. (C, D) Representative movement cytometry plots displaying the result of knockdown on cell surface area manifestation of ICAM-1, E-selectin, and P-selectin. HUVECs transfected with 25 nM siRNA or control were treated or not treated with TNF-.
