Figure 4

Downregulation of Sirt6 results in increased susceptibility of mouse colonic epithelial cells to cell death challenges, whereas Sirt6 overexpression increases resistance of mouse colonic epithelial cells to cell death challenges. (A) Silencing of Sirt6 expression in colonic epithelial cells was achieved using siRNA technology. The Sirt6 knockdown in vitro was confirmed using qRT-PCR (upper panel) and Western blot (lower panel). (B) Silencing of Sirt6 increases susceptibility of colonic epithelial cells to cell death challenges. YAMC cells were subjected to transfection with siSirt6 or siControl, as described above. Forty-eight hours later, cells were treated with indicated treatments. Twenty-four hours after being challenged with cell death inducers, cell viability was assessed using CellTiter-Glo assay. The experiments were performed three times. Results are expressed as mean ± SEM. n = 3 in each group. *, P < 0.05 versus control; **, P < 0.01 versus control. (C) Ectopic expression of Sirt6 in intestinal epithelial cells by transfection with pSirt6 (ie, pIRES2-zsGreen1-mSirt6 plasmid). Sirt6 overexpression was confirmed with Western blot. pVector, the pIRES2-zsGreen1 plasmid (ie, control). (D) Overexpression of Sirt6 renders colonic epithelial cells resistant to cell death challenges. YAMC cells were subjected to transfection with pSirt6 or pVector, as described in panel C. Forty-eight hours later, they were subjected to indicated treatments. Twenty-four hours later, cell viability was assessed using CellTiter-Glo assay. Results are expressed as mean ± SEM and represent average of findings from three independent experiments. n = 3 in each group. *, P < 0.05 versus control; **, P < 0.01 versus control.