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 7. SIRT6 knockdown does not directly induce DNA damage at the times when the cell cycle is inhibited. (A) Representative images for the effect of SIRT6 knockdown on the formation of DNA damage foci. HUVECs were transfected with 25 nM control or SIRT6 siRNA. H2O2 (200 μm) and etoposide (10 μm) were used as positive controls to induce DNA damage. After 3 d, cells were stained with anti-γ-H2AX and anti-53BP1 antibodies. DAPI was used to stain nuclei. Scale bars represent 20 μm. (B) Quantification of DNA-damaged cells. Cells with more than ten γ-H2AX or five 53BP1 foci were scored. Data are expressed as the mean ± SD (n = 3). *P < 0.05 vs. control siRNA. **P < 0.001 vs. control siRNA. (C) Comet images of HUVECs treated with control or SIRT6 siRNA. HUVECs were treated with 25 nM control or SIRT6 siRNA. After 3 d, dissociated single cells were subjected to alkaline comet assay. Cells treated with H2O2 for 1 h were used as positive control. (D) Analysis of comet images. Percent DNA in the tail and tail moment of damaged cells were quantified using OpenComet. Data are expressed as the mean ± SD (n = 3). *P < 0.05 vs. control siRNA. **P < 0.01 vs. control siRNA.