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. Author manuscript; available in PMC: 2012 Jun 4.
Published in final edited form as: Cell. 2007 Sep 21;130(6):1095–1107. doi: 10.1016/j.cell.2007.07.035

Figure 3. Nampt-Mediated Protection against Genotoxicity Requires SIRT3 and SIRT4.

Figure 3

(A) Survival of HEK293 cells stably expressing Nampt following exposure to MMS in the presence or absence of the SIRT1-specific inhibitor EX-527.

(B) siRNA knockdown of SIRT1 using a pool of four siRNA oligos, compared to nontargeting siRNA controls. Cells were cotransfected with FAM-tagged fluorescent oligos and percentage cell death was determined by FACS as a ratio of PI/FAM-positive cells versus total FAM-positive cells.

(C) HEK293 cells were treated with 100 μM sirtinol, a pan sirtuin inhibitor. All experiments were carried out three times in triplicate.

(D and E) SIRT3 or SIRT4 was knocked down in HEK293 cells stably overexpressing Nampt using pools of specific siRNA oligos, and cells were then treated with MMS and scored for survival.

(F) Cells from (D) were probed by western blotting for cleaved caspase-3, an indicator of apoptosis.

(G) Immunoprecipitation (IP) of AceCS2 from cell lysates of control and Nampt-overexpressing HEK293 cells transfected with control vector or AceCS2-HA for 48 hr. The levels of acetylated AceCS2 in IPs were analyzed by western blotting.

Bars represent the mean of three experiments ± SD.