Figures & data
Figure 1 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.
Figure 2 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.
Figure 3 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.
Figure 4 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.
