Priority Research Paper Volume 12, Issue 21 pp 20946—20967

Sirtuin 6 deficiency induces endothelial cell senescence via downregulation of forkhead box M1 expression

Figure 3. Downregulated expression of SIRT6 induces endothelial cell dysfunction. (A) Effect of SIRT6 siRNA on in vitro tube formation in HUVECs. HUVECs transfected with 25 nM of the indicated siRNA were cultured on Matrigel to check in vitro angiogenesis activity of endothelial cells. The representative micrographs of tube formation in HUVECs. (B) Western blot analysis showing the effect of SIRT6 siRNA on the expression of eNOS and KLF2. β-Actin was used as a loading control. (C, D) Representative flow cytometry plots showing the effect of SIRT6 knockdown on cell surface expression of ICAM-1, E-selectin, and P-selectin. HUVECs transfected with 25 nM control or SIRT6 siRNA were treated or not treated with TNF-α (50 ng/mL) for 4 h. Cells were stained with the fluorochrome-conjugated antibodies and analyzed by flow cytometry. (E) Graphs showing ICAM-1, E-selectin, and P-selectin expression levels in the cells. Data were obtained by analyzing the mean fluorescence intensity of each inflammatory molecule on HUVECs, which were differentially treated with control or SIRT6 siRNA in the absence and presence of TNF-α. *P < 0.01 vs. control siRNA. #P < 0.01 vs. control siRNA with TNF-α.