Fig 2: SIRT6 deletion affects gene expression in the brain. (A) Experimental design: RNA was isolated from the brains of three SIRT6-KO mice, three age-matched WT mice (21 days), and three old WT mice (22-26 months). (B) Hierarchical clustering of samples. Colors represent Pearson correlation coefficients. (C) Expression levels of genes identified as upregulated or downregulated in a comparison between SIRT6-KO and young WT mice. Red color corresponds to increased expression, while blue color corresponds to decreased expression. (D) Expression levels of genes identified as upregulated or downregulated in a pool of SIRT6-KO and old WT mice compared to young WT mice. Colors are as in (C).

Fig 3: Dynamic change of SIRT6 expression is related to APAP injury-responsive liver repair. (A) Male C57BL/6 mice were administered a single dose of 400 mg/kg APAP by intraperitoneal injection and killed at 0, 6, 12, 24, 48 and 72 h after APAP treatment (n = 6). (B) H&E-stained liver sections, (C) serum ALT and (D) AST activities from APAP-treated mice over a time course of 0–72 h after 400 mg/kg APAP challenge (n = 6). (E) Western blot analysis of total and nuclear SIRT6 protein expression in APAP-treated mice at various time points after APAP challenge. (F) Densitometric analysis of Western blots (n = 3−4). Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 versus 0 h.

Fig 4: NFATc4 participated in the anti-hypertrophic effect of SIRT6. (A–C) Western blot analysis of the protein expression of SIRT6, NFATc4, and BNP in NRCMs transfected with or without WT-NFATc4, WT-SIRT6, and treated with 100 μM PE for 12 h. (D–F) The protein expression of SIRT6, NFATc4, and BNP in NRCMs transfected with siRNAs of SIRT6, NFATc4, or negative control (NC) for 48 h. The data were presented as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. Vector or NC. #P < 0.05, ###P < 0.001 vs. PE or si-SIRT6. $P < 0.05, $$P < 0.01, vs. WT-SIRT6 + PE or si-NFATc4. n = 4.

Fig 5: The SIRT6-mediated EZH2/FOXC1 pathway is critical for the beneficial effects of MDL-811 in LPS-induced neuroinflammatory mice model and brain ischemic stroke mice model. (A) Western blot analysis on the protein expression of FOXC1 and H3K27me3 in the cerebral cortex region of the brain of mice at 3 h after treatment of LPS (n = 4). Results were normalized to H3 or β-actin. Data are representative of three independent experiments. (B) Acetylated EZH2 levels in the cerebral cortex region of mice at 3 h after treatment of LPS (n = 4). IgG is the negative control and β-actin is the input loading control. Data are representative of three independent experiments. (1) Control; (2) LPS+vehicle; (3) LPS+MDL-811 (1 mg/kg); (4) LPS+MDL-811 (10 mg/kg). (C) Statistical analysis of the immunoblots in (A) and (B). Results are expressed as mean ± SEM; ∗P < 0.05, ∗∗∗P < 0.001 versus control group; #P < 0.05, ##P < 0.01, ###P < 0.001 versus vehicle group. (D) Western blot analysis on the protein expression of FOXC1 and H3K27me3 in the peri-infarct cerebral cortex region of the brain of mice at 72 h post tMCAO treated with MDL-811 (n = 4). Data are representative of three independent experiments. (E) Acetylated EZH2 levels in the peri-infarct cerebral cortex region of mice at 72 h post tMCAO treated with MDL-811 (n = 4). IgG is the negative control and β-actin is the input loading control. Data are representative of three independent experiments. (1) Sham; (2) tMCAO+vehicle; (3) tMCAO+MDL-811 (1 mg/kg); (4) tMCAO+MDL-811 (10 mg/kg). (F) Statistical analysis of the immunoblots in (D) and (E). Results are expressed as mean ± SEM; ∗P < 0.05, ∗∗∗P < 0.001 versus sham group; #P < 0.05, ##P < 0.01, ###P < 0.001 versus the vehicle group.