SIRT6 overexpression is selectively cytotoxic to multiple cancer cell lines. (A) The indicated non-cancerous and cancer cell lines were transfected with a SIRT6-expressing vector or a control plamsid. Representative immunoblot of SIRT6 levels 24 h after transfection. SIRT6 expression is similar in cancer and normal cells. (B) Represenative images of cells taken 72 h post-transfection. (C) Quantification of SIRT6 toxicity. Cell survival 72 h post-transfection, measured as the number of adherent cells transfected with SIRT6 expressing vector compared to the number of adherent cells transfected with a control plasmid. (D) Quantification of SIRT6-induced apoptosis. Relative levels of apoptosis 72 h post-transfection, determined by Annexin V staining. Experiments were repeated three times and error bars show SD.

SIRT6 induces apoptosis via the p53 and p73 cell death pathways. (A) HeLa cells were transfected with plasmids encoding p53 dominant-negative fragment (p53DN) or p73 dominant-negative fragment (p73DN). Blockade of p73 signaling is sufficient to prevent SIRT6-induced apoptosis. (B) HT1080 cells were transfected with indicated expressing plasmids. Inhibition of both p53 and p73 signaling is required to prevent SIRT6-induced apoptosis. Experiments were repeated three times, and error bars show SD.

SIRT6 mono-ADP-ribosyltransferase activity is required for killing cancer cells. (A) Adherent cell count 72 h after transfection with indicated SIRT6-encoding vector relative to a control plasmid. (B) Quantification of apoptosis levels 72 h after transfection with a plasmid expressing wild-type SIRT6 or its mutants. (C) Pretreatment of HT1080 cells with the PARP1 inhibitor PJ-34 did not prevent SIRT6-induced apoptosis. (D) Overexpresison of PARP1 did not induce apoptosis in HT1080 cells. Experiments were repeated three times, and error bars show SD.

SIRT6 induces apoptosis in cancer cells via ATM pathway. (A) Cancer cells were pretreated with DMSO or ATM inhibitor wortmannin prior to transfection with the SIRT6-encoding vector or a control plasmid. Pretreatment with ATM inhibitor attenuated SIRT6-mediated apoptosis. (B) Glucose intake was measured in cancer cells. Transfection with SIRT6-encoding vector did not alter the kinetics of glucose intake compared to control. (C) Glucose depletion from the media was measured in cancer cells transfected with the SIRT6-encoding vector or a control plasmid. No significant changes were observed within 24 h of transfection. Experiments were repeated three times, and error bars show SD.