Fig 2: SIRT6 is required to recruit CHD4 to chromatin following DNA damage. (A) HCT116 cells were treated with 1 µM DOX/40 µM VP16 /10 µM cisplatin/0.5% BSA for 0.5 h or treated with 200 J/m2 UV. The cells were fractionated (Dt/Chr) and analyzed by western blotting. (B) DR-U2OS cells were transfected with an I-SceI expression plasmid for up to 48 h. ChIP experiments were performed using an anti-SIRT6 or an anti-CHD4 antibody. SIRT6 and CHD4 binding to the DNA sequences flanking the I-SceI sites were measured. IgG was used as a negative control. Data are normalized to the control (no I-SceI) samples. (C) HCT116 cells were transfected with NS (non-specific) or two independent SIRT6 specific siRNAs for 48 h in the presence or absence of 40 µM VP16. Chromatin fractions were extracted and analyzed by western blotting. (D) DR-U2OS cells were transfected with NS or SIRT6-specific siRNA for 48 h; then, the cells were transfected with or without an I-SceI expression plasmid for 24 h. ChIP experiments were performed using an anti-CHD4 antibody. CHD4 binding to the DNA sequences flanking the I-SceI sites was measured. (E) HCT116 cells were transfected with NS or two independent CHD4-specific siRNAs for 48 h in the presence or absence of 40 µM VP16. Chromatin fractions were extracted and analyzed by western blotting. (F) DR-U2OS cells were transfected with or without the I-SceI expression plasmid for 48 h. Sequential ChIP-reChIP was performed on the I-SceI DNA damage sites by immunoprecipitation with an anti-SIRT6 antibody followed by immunoprecipitation with an anti-CHD4 antibody. The data represent the means ± SEM (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001).

Fig 3: SIRT6 is a p53 co-activator and recruits RNA polymerase II (RNAP II) to the CDS1 and CDS2 promoters in response to palmitic acid (PA) treatmenta HCT116 cells were treated with or without 0.2 mM PA for 18 h and proteins were extracted for co-immunoprecipitation with an anti-SIRT6 antibody, followed by western blotting with an anti-SIRT6 or anti-RNAP II antibody to detect the endogenous interaction between RNAP II and SIRT6. b HCT116 and HCT116 (p53-/-) cells were treated with 0.2 mM PA for 18 h and proteins were extracted for co-immunoprecipitation with an anti-SIRT6 antibody, followed by western blotting with an anti-SIRT6 or anti-RNAP II antibody to detect the endogenous interaction between RNAP II and SIRT6. c, d HCT116 cells were transfected with a SIRT6 siRNA or a non-specific siRNA negative control (NC) and then harvested for a ChIP assay to detect enrichment of RNAP II around the CDS1 and CDS2 promoters. The bands containing anti-immunoglobulin G (IgG) served as negative controls. The bar (-) represents the means (n = 3). e A schematic showing a possible mechanism by which p53 coordinates with SIRT6 to regulate the expression of cardiolipin de novo biosynthesis-related genes. In the presence of PA, p53 recruits SIRT6 to bind chromatin in a direct complex. Here, SIRT6 recruits RNA polymerase II to the CDS1 and CDS2 promoters to activate their transcription and permit CL biosynthesis. IB immunoblot, IP immunoprecipitation

Fig 4: Sirt6 heterozygous deletion increases tumor latency and enhances survival in the MMTV-PyMT mammary tumor model. a Tumor masses were excised from MMTV-PyMT+/-; Sirt6+/+ and MMTV-PyMT+/-; Sirt6+/- mice; protein lysates were generated, and Sirt6 and vinculin levels were detected by Western blot. b Tumor latency (upper panel) and overall survival (lower panel) of MMTV-PyMT+/-; Sirt6+/+ (n = 39) and of MMTV-PyMT+/-; Sirt6+/- (n = 30) mice were monitored over time. c Histopathological analysis of the mammary glands from MMTV-PyMT+/-; Sirt6+/+ and MMTV-PyMT+/-; Sirt6+/- mice (hematoxylin-eosin; × 40 and × 400) shows poorly differentiated (G3) murine ductal carcinoma of the breast consisting in dysmetrical elements with solid growth, brisk mitotic activity, and scattered areas of necrosis in both types of tumor. d Mammary glands from 4-week-old MMTV-PyMT+/-; Sirt6+/+ (n = 3) and MMTV-PyMT+/-; Sirt6+/- (n = 3) mice were stained by carmine alum (left) and used to quantify the TEB (right). LN, lymph node. Data are presented as mean ± SD; ns, not statistically significant. e Blood glucose levels in Sirt6+/+ (n = 6), Sirt6+/- (n = 3), MMTV-PyMT+/-; Sirt6+/+ (n = 3), and MMTV-PyMT+/-; Sirt6+/- (n = 6) mice. Data are presented as mean ± SD. ns, not statistically significant

Fig 5: SIRT6 knockdown inhibits proliferation, migration and invasion of ccRCC cells. (A and B) The results of the Cell Couting Kit-8 assay. ccRCC-derived 769-P and 786-O cells were transfected with si-NC or si-SIRT6 (si-SIRT6#1 and si-SIRT6#2). Transfection efficiency was detected via western blot analysis (insets). ß-actin was used as the loading control. (C) The results of the colony formation assay. ccRCC-derived 769-P and 786-O cells were transfected with si-NC or si-SIRT6 (si-SIRT6#1 and si-SIRT6#2). Cells were subjected to (D) migration (E) invasion assays 24 h post-transfection. The results were analyzed using ImageJ software and expressed in histograms. Data are presented as the mean ± standard deviation (n=3). *P<0.05, **P<0.01, ***P<0.001. SIRT6, sirtuin 6; ccRCC, clear cell renal cell carcinoma; si, small interfering; NC, negative control.